Regulation of Oct4 Expression by Jun-N Terminal Kinase/Cjun

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REGULATION OF OCT4 EXPRESSION BY JUN-N TERMINAL KINASE/CJUN

SIGNALING IN MURINE EMBRYONIC STEM CELLS

Manal Hosawi

A Thesis Presented to

The Faculty of Humboldt State University

In Partial Fulfillment of the Requirements for the Degree

Master of Science in Biology

Committee Membership

Dr. Amy Sprowles, Major Professor, Committee Chair

Dr. Bruce A. O’Gara, Committee Member

Dr. Edward Metz, Committee Member

Dr. Jacob P. Varkey, Committee Member

Dr. Erik S. Jules, Graduate Coordinator

May 2016

ABSTRACT

REGULATION OF OCT4 EXPRESSION BY JUN-N TERMINAL KINASE/CJUN SIGNALING IN MURINE EMBRYONIC STEM CELLS

Manal Hosawi

Oct4 is a POU class V transcription factor that functions to maintain the

pluripotent state of embryonic stem cells by activating or repressing the transcription of

hundreds of target genes (Boyer et al. 2005). Aberrant expression of Oct4 has been

reported in a number of cancers, suggesting that it may also play a role in cellular

transformation (Tai et al. 2005). This study examines the role of the proto-oncogene cJun

and its upstream kinase Jun N terminal kinase (JNK) in the regulation of Oct4 expression

and the pluripotent state. Our laboratory identified a putative AP-1 binding site approximately 2500 bp upstream of the Oct4A transcription start site. We modulated the cJun/JNK pathway in two murine embryonic stem cell lines through chemical treatments and transient transfection of wild type and mutant GFP cJun constructs. Our results show

mESCs overexpressing cJun forms or treated with 10 ng/ml, 50 ng/ml anisomycin and 50

µM JNK inhibitor SP600125 have an increase in the Oct4A isoform by

immunocytochemistry and Western blot analysis, but not the total amount of Oct4. The

potency of these cells is also affected. Increased JNK/cJun activity increased in alkaline

phosphatase activity but resulted in a significant reduction of pancreatic islet like cluster

size, number, insulin expression and the neuronal network between clusters. Loss of

JNK/cJun activity had the opposite result: transfection of the transcriptionally inactive

ii cJun mutant L40/42A or treatment with SP600125 resulted in increased number, size and neuronal network formation among the pancreatic islet like clusters. JNK modulation by chemical treatment also affected the formation of cardiomyocyte beating clusters. These results indicate the importance of the regulation of JNK/cJun pathway in stem cell potency, potentially through changes in the expression level of Oct4A. Future studies exploring the role of Oct4A in pluripotency and JNK/cJun activity in early embryonic development could provide insight into the role of this isoform in cellular transformation.

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ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my advisor Dr. Amy Sprowles who provided continues support during my Master study at Humboldt State University.

Her motivation, patience and guidance helped me in all the time of research and writing thesis. I could not have imagined having a better advisor for my Master study. I also would like to thank my thesis committee members: Dr. Bruce A. O’Gara, Dr. Edward

Metz, and Dr. Jacob P. Varkey, for their insightful comments, patience and advice.

My sincere thanks also goes to my lab mates in Dr. Sprowles laboratory, Ms.

Abigail Petersen, Ms. Akela Kuwahara, Ms. Haley E. Du Bois, Mr. Jack Guccione, Ms.

Jacqueline Trzeciak, Mr. Johnny L. Castillo, Mr. Christopher H. Mendoza, Ms. Lauren

Dahl and Mr. Michael Lopez, for their assistance and support.

I want to express my special gratitude to my country Saudi Arabia for financial assistance during my stay in the United States and providing me with this opportunity to study abroad. I especially would like to express my eternal gratitude to my family; my father Mr. Mosa Hosawi, my mother Mrs. Sanaa Husayn and my brothers, for the endless support, encouragement, and love during my graduate education and throughout life. To my family, thank you for encouraging me in all of my pursuits and inspiring me to follow my dreams.

TABLE OF CONTENTS

REGULATION OF OCT4 EXPRESSION BY JUN-N TERMINAL KINASE/CJUN SIGNALING IN MURINE EMBRYONIC STEM CELLS ...... i ABSTRACT ...... ii REGULATION OF OCT4 EXPRESSION BY JUN-N TERMINAL KINASE/CJUN SIGNALING IN MURINE EMBRYONIC STEM CELLS ...... ii ACKNOWLEDGEMENTS ...... iv TABLE OF CONTENTS ...... v LIST OF TABLES ...... vii LIST OF FIGURES ...... viii LIST OF APPENDICES ...... xi INTRODUCTION ...... 1 Transcription Factor Oct4 ...... 5 The Regulation of Oct4 Expression ...... 6 The Transcription Factor cJun ...... 9 Biological Roles of the Transcription Factor cJun and JNK ...... 9 Specific Aims: ...... 13 1. Determining the role of cJun activity on Oct4 expression in murine embryonic stem cells...... 13 2. Evaluating the role of cJun signaling on mESC potency...... 14 3. Evaluating the role of JNK signaling on mESC potency...... 14 MATERIALS AND METHODS ...... 15 293T Cell Culture ...... 15 J1 and B6/BLU Murine Embryonic Stem Cell Culture ...... 16 Chemical Treatment of Mouse Embryonic Stem Cells and 293T ...... 17 Protein Extraction and Western Blot ...... 17 Immunocytochemistry ...... 20 Transient Transfection of 293T Cells ...... 21 Transient Transfection of Mouse Embryonic Stem Cells ...... 22 Differentiation of Mouse Embryonic Stem Cells ...... 23 Embyoid Body formation ...... 23 Directed differentiation from EBs to beating cardiomyocytes ...... 24 Directed differentiation from EBs to Pancreatic islet-like insulin secreting clusters 24 Alkaline phosphatase ...... 25 Statistical Analysis ...... 26

RESULTS ...... 27 The Role of the JNK/cJun Pathway on Oct4 Expression in Murine Embryonic Stem Cells ...... 27 Examining the modulation of JNK/cJun pathway by chemical treatment in 293T .. 27 Examining the modulation of JNK/cJun pathway by chemical treatment on Oct4 expression in mESCs ...... 35 Testing the Overexpression of cJun on Oct4 ...... 55 The Role of CJun Signaling on Murine Embryonic Stem Cell Potency...... 69 Testing the effects of cJun on alkaline phosphatase activity ...... 69 Examining the effects of cJun on directed differentiation ...... 72 Directed differentiation into Pancreatic Islet Like Clusters and Cardiomyocytes .... 72 The Role of JNK Signaling on Murine Embryonic Stem Cell Potency ...... 82 Testing the effects of modulating JNK signaling on alkaline phosphatase activity . 82 Testing the effects of modulating JNK on the differentiation of mESCs ...... 87 Directed differentiation into Pancreatic islet like clusters ...... 87 Directed differentiation into Cardiomyocytes ...... 98 DISCUSSION ...... 101 CJun and JNK Signaling Affects Oct4 Expression...... 101 CJun/JNK Activity Effects MESC Potency ...... 102 Differences in the Subcellular Localization of CJun Between 293T and MESCs ..... 106 Conclusions ...... 108 LITERATURE CITED ...... 110

LIST OF TABLES

Table 1. Treatment with SP600125 at 10 μM and anisomycin at 10 ng/ml did not change the viability of mESCs...... 37 Table 2. The addition of anisomycin at 50 ng/ml resulted in increased nuclear localization of JNK in mESCs...... 41 Table 3. The addition of anisomycin at 50 ng/ml resulted in cytoplasmic and nuclear localization of p-cJun S63...... 47 Table 4. Comparison of the subcellular localization and expression of p-cJun S63 between 293T and mouse embryonic stem cells with the addition of SP600125 and anisomycin...... 49 Table 5. Comparison of the subcellular localization and expression of p-cJun S73 between 293T and mouse embryonic stem cells post the addition of SP600125 and anisomycin...... 50 Table 6. Differences in total number, size and network formation in pancreatic Islet-Like clusters post the addition of different forms of cJun...... 81 Table 7. Differences in total number, size and network formation in pancreatic Islet-Like clusters derived from mESCs treated with SP600125 or anisomycin...... 96 Table 8. Treatment with SP600125 showed the highest number of beating cardiomyocytes...... 98

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LIST OF FIGURES

Figure 1. The development of mouse embryo from a morula to Egg cylinder stage...... 4 Figure 2. The identification of AP-1 sites in the regulatory sequences of Mus musculus (mouse)...... 8 Figure 3. Chemical modulation of the JNK pathway affects the levels and subcellular localization of cJun in 293T cells...... 29 Figure 4. Modulating the JNK pathway by chemical treatment affects the levels and the subcellular localization of p-cJun 63 in 293T cells...... 30 Figure 5. Modulating the JNK pathway by chemical treatment affects the levels of p-cJun S73 in 293T cells...... 31 Figure 6. p-JNK expression levels in 293T cells treated with anisomycin or SP600125. 32 Figure 7. Modulating JNK pathway by chemical treatments affects levels of p-cJun in 293T cells...... 34 Figure 8. Identification of the optimal concentration of anisomycin and SP600125 in J1 mESCs...... 36 Figure 9. SP600125 ± LIF reduces the number of J1 mouse embryonic stem cells. Cells were under treatment for 6 days...... 38 Figure 10. Anisomycin at 50 ng/ml resulted in increases the nuclear localization of JNK...... 40 Figure 11. Modulating JNK pathway by chemical treatments for 2 and 4 hours showed increase p-JNK in the nuclear fraction in mESCs...... 42 Figure 12. Modulating JNK/cJun pathway by chemical treatments affects the subcellular localization of p-cJun S63 in mESCs...... 45 Figure 13. . Modulating JNK/cJun pathway by chemical treatments showed nuclear subcellular localization of p-cJun S73 in mESCs...... 46 Figure 14. . Modulating JNK pathway by chemical treatments for 2 and 4 hours affects the subcellular localization of p-cJun and the level of p-cJun in mESCs...... 48 Figure 15. . Different subcellular localization of c-Jun and phospho c-Jun among untreated 293T and mESCs ...... 51 Figure 16. Oct4A expression is increased in mESCs treated with 50 and 10 ng/ml Anisomycin or 50 μM SP600125...... 53 Figure 17. Modulating JNK pathway by chemical treatments for 2 and 4 hours affects the level of Oct4A in the nuclear fraction of mESCs...... 54 Figure 18. Expression of GFP cJun, GFP cJun R54A and GFP cJun L40/42A is highest 36 hours post transfection of J1 mESCs...... 58 Figure 19. Selection of J1 mESCs transiently expressing GFP cJun, GFP cJun R54A or GFP cJun L40/42A did not induce stable expression of the cJun fusion proteins.. .. 59 Figure 20. GFP cJun and GFP cJun mutants are expressed in J1 murine embryonic stem cells and predominantly localized to the nucleus...... 62 Figure 21. GFP cJun expression in J1 embryonic stem cells did not show a measurable increase in total Oct4 protein...... 63

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Figure 22. Oct4A expression is increased in J1 embryonic stem cells expressing GFP cJun or GFP cJun variants but not in GFP alone...... 64 Figure 23. Transfected J1 cells with cJun forms show statistically significant increase in Oct4A expression...... 65 Figure 24. GFP cJun, GFP cJun, GFP cJun L40/42A and GFP cJun R54A decreased alkaline phosphatase activity in J1 murine embryonic stem cells...... 71 Figure 25. Embryoid body formation is not affected by transient transfection of GFP cJun constructs...... 73 Figure 26. Decrease in total number, size and network formation of Pancreatic Islet-like clusters in mouse embryonic stem cells transfected with wild-type cJun...... 76 Figure 27. Differences in alpha tubulin and total number of Pancreatic Islet-like clusters derived from J1 mouse embryonic stem cells transfected with wild type GFP cJun...... 77 Figure 28. Transient Expression of GFP cJun in murine embryonic stem cells reduces the number of pancreatic islet like clusters...... 78 Figure 29. Transient expression of wild type GFP cJun in murine embryonic stem cells resulted in significant decrease in the size of pancreatic islet like cluster...... 79 Figure 30. Transient expression of wild type GFP cJun in murine embryonic stem cells resulted in significant decrease of network formation between clusters in comparison to GFP cJun L40/42A and GFP genotypes...... 80 Figure 31. SP600125 treatment of murine embryonic stem cells resulted in the highest formation of undifferentiated mouse embryonic stem cells colonies...... 84 Figure 32. Treatment of embryonic stem cells with SP600125 ± LIF resulted in the highest undifferentiated mouse embryonic stem cells colonies 6 days post treatment...... 85 Figure 33. Mouse embryonic stem cells morphology is maintained after 4 days of 10 μM SP600125 ± LIF treatments...... 86 Figure 34. Treatment of J1 murine embryonic stem cells with anisomycin or SP600125 affects the embryoid body morphology in J1cell line...... 88 Figure 35. Treatment of J1 murine embryonic stem cells with anisomycin or SP600125 affects the amount of circular and clumped embryoid bodies...... 88 Figure 36. Pancreatic Islet-like clusters derived from J1 murine embryonic stem cells treated with 10 ng/ml anisomycin at or 10 µM SP600125 show different levels of insulin expression and neuronal morphology...... 89 Figure 37. Chemical modulation of JNK activity in J1 mESCs affects pancreatic islet like cluster formation...... 91 Figure 38. Chemical modulation of JNK activity in B6/BLU mESCs affects pancreatic islet like cluster formation...... 92 Figure 39. Chemical modulation of JNK activity in murine embryonic stem cells affects the total number of pancreatic Islet- Like clusters...... 93 Figure 40. Chemical modulation of JNK activity in murine embryonic stem cells affects the size of pancreatic Islet-Like clusters...... 94

Figure 41. Chemical modulation of JNK activity in murine embryonic stem cells affects neuronal network formation between pancreatic Islet-Like clusters...... 95 Figure 42. . J1 mESCs can produce insulin secreting pancreatic islet like clusters after chemical modulation of JNK activity...... 97 Figure 43. Murine embryonic stem cells treated with SP600125 ± LIF for 2 days successfully formed Embryoid body...... 99 Figure 44. Murine embryonic stem cells treated with 10 µM SP600125 ± LIF successfully formed cardiomyocyte beating colonies...... 100

LIST OF APPENDICES

APPENDIX A. GENERATION of pLVX-EF1α-AcGFP1-CJUN CONSTRUCTS. .... 120 APPENDIX B...... 144

xi 1

INTRODUCTION

Understanding the regulation of the pluripotent state is an important step for

understanding cell fate and early embryonic development. It can also enhance our ability

to diagnose and potentially treat a myriad of human disorders and diseases (Reviewed in

Reya et al. 2001; reviewed in Pardal et al. 2003; reviewed in Sell, 2004). Embryonic stem

cell cultures are a system of choice for studying these processes. First derived in 1981

(Evans and Kaufman, 1981; Martin, 1981), these cultures are defined by three unique

properties: self-renewal, increased proliferation rates and pluripotency (Figure 1).

Self-renewal ensures the maintenance of the stem cell population through cell

division. In tissue resident stem cells this is achieved through asymmetric cell division

result in different kinds of daughter cells: one that is identical to the original stem cell,

and another daughter cell with limited proliferative capacity. These daughter cells then

differentiate into specific cell types and become more specialized (Lajtha, 1979; Potten

and Lajtha, 1982). When cared for correctly, embryonic stem cell cultures divide

symmetrically to expand the total population. This happens rapidly, as embryonic stem

cells exhibit increased proliferation rates due to their unique and controlled cell cycle

structure: short G1 phase and a longer S-phase. This is believed to be due in part to reduced heterochromatin formation in these cells. (Reviewed in White and Dalton, 2005; reviewed in Neganova and Lako, 2008).

Pluripotency refers to the ability of embryonic stem cells to differentiate into all cells of the organism. For an embryonic stem cell culture to be considered pluripotent, it

2 must have the ability to differentiate into cells derived from each of the three germ layers:

mesoderm, endoderm and ectoderm. Maintenance of the pluripotency state of murine

embryonic stem cells (mESCs) in vitro is traditionally achieved by culturing the cells as

aggregates in the presence of the self-renewal signals provided by the leukemia inhibitory

factor (LIF) and a fibroblast feeder layer. The absence of those signals initiates the

differentiation of the ESCs by forming embryonic bodies (EBs) that spontaneously form

the three primary germ layers in vitro. EBs may be directed to differentiate into a variety

of specialized cell types including cardiac and skeletal muscle as well as hematopoietic,

pancreatic, hepatic, lipid, cartilage, or neuronal and glial cells (Wobus et al. 1984; Wobus

et al. 1997; Choi et al. 2002; Wobus et al. 2002).

The co-expression of three genes is essential for maintaining the pluripotent state:

Oct4, NANOG, and SOX2 (Reviewed in Loh et al. 2006; Masui et al. 2007). Multiple

studies have shown modulating the expression of these genes leads to changes in cell fate

and embryonic development. The absence of Oct4 and SOX2 at early stages results in

early embryonic lethality, impairing the epiblast formation and trophectoderm formation

(Niwa et al. 2000; Avilion et al. 2003). The loss of Nanog in the inner cell mass (ICM)

results in impairing the formation of the epiblast and forming parietal endoderm-like

cells. Yet, the absence of Nanog in ES cells causes loss of pluripotency and differentiation into extra embryonic endoderm-like cells (Mitsui et al. 2003).

Pluripotency genes are sometimes overexpressed in cancer cells, as noted in a study that compared reported significant overexpression of Oct3/4 and SOX2 between 40 human tumors when compared to their counterpart normal tissues (Schooenhals et al.

3 2009). Other studies have also showed the presence of Oct4, SOX2 and Nanog

expression in gastric cancers and primary mediastinal germ cell tumors (Liu et al. 2010;

Matsuoka et al. 2012). Studies such as these support the cancer stem cell hypothesis. First

described by Reya et al in 2001, it suggests a population of stem cell-like cells that has

some of stem cells proprieties including the expression of pluripotency. Furthermore,

studies show cancers to arise from cancer stem cells that capable of self-renewal and forming tumor heterogeneity (Quintana et al. 2008; Visvader and Lindeman, 2008).

Figure 1. The development of mouse embryo from a morula to Egg cylinder stage. The pluripotent lineages and differentiation patterns for mouse embryo (Niwa 2007).

Transcription Factor Oct4

Oct4 is a POU class V transcription factor expressed in the totipotent and pluripotent stem cells of the pregastrulation embryo. It functions to maintain the pluripotent state of embryonic stem cells by activating or repressing the transcription of hundreds of target genes (Niwa et al. 2000; Boyer et al. 2005). Oct4 is a preeminent transcription factor in the induction of induced pluripotent stem cells (IPSs) (Takahashi and Yamanaka, 2006). It has also been found to be highly expressed in cancers such as human glioma, breast and seminoma carcinoma (Uche et al. 2005; Weiren et al. 2013).

The expression level of Oct4 is strictly regulated. The very early expression of

Oct4 is dictated by the maternally inherited mRNA present in the cytoplasm of the female egg before fertilization (Rosner et al.1990; Pesce and Schöler, 2001). Modulation of Oct4 affects hESCs and mESCs differently and results in distinct differentiation patterns between the two cell types. Overexpression of Oct4 in mESCs increases expression of endoderm and mesodermal markers (Niwa et al. 2000; Zeineddine et al. 2006) and down regulation of Oct4 expression by RNA interference resulted in the initiation of trophectodermal differentiation (Velkey et al. 2003). Overexpression of Oct4 in hESCs results in promoting the differentiation of endoderm, while the down regulation of Oct4 initiates mesoderm and endoderm differentiation (Rodriguez et al. 2007). The expression of Oct4 and other pluripotency genes are down regulated when the ESCs begin to differentiate (Loh et al. 2006; Masui et al. 2007). The inhibition of Oct4 expression in the developing embryo, in cultured human embryonic stem cells (hESCs), mouse embryonic

6 stem cells (mESCs), or in embryonal carcinoma cells (ECs) lead to the loss of the pluripotency (Nichols et al. 1998; Matin et al. 2004).

The Regulation of Oct4 Expression

Oct4 gene expression is controlled by various mechanisms, including transcription factor binding, epigenetic modification, and alternative splicing. Three promoter elements have been identified that regulate the transcription of Oct4. They include proximal enhancer (PE), a proximal promoter (PP) located approximately 1.2 Kb upstream of the transcription start site, and a distal enhancer (DE) located approximately

3.3 kb upstream of the murine Oct 3/4 transcription start site. These elements provide sites for the transcription factors and regulatory proteins to regulate the expression of

Oct4 in undifferentiated stem cells, ES, and EC cells (Okazawa et al. 1991; Minucci et al.

1996; Yeom et al. 1996; Okumura et al. 2005). The combination of regulatory proteins at the three elements in Oct4 promoter depends on the development stage of the embryo.

The PE regulates the Oct4 expression in epiblast cells while the DE regulates the Oct4 expression in the inner cell mass, germ cell precursors and ES cells (Pesce and Scholer,

2001; Chambers and Smith, 2004; Boiani and Scholer, 2005). In the mouse Oct4 gene, the DE is composed of two functional elements (2A at –2080/–2070 and 2B at -2040/-

2024), which are required for proper Oct4 expression in pluripotent cells (Okumura et al.

2005). These sequences are highly conserved among human, bovine and mouse

(Nordhoff et al. 2001).

7 Scientists have identified multiple transcription factors that activate or inhibit the

Oct4 expression by binding at either PP, DE or PE. Recent evidence implicates the canonical Wnt/ β - catenin cascade in the regulation of Oct4. An LEF/TCF binding site was recently described at -2043 and -875 upstream of the Oct4 promoter and shown to modulate the expression of the Oct4 gene (Li et al. 2012). Bioinformatics analysis of the

Oct4 promoter in our laboratory showed the presence of an AP-1site at the DE (bp -2257) of the murine Oct4 promoter (Figure 2). Others were identified in introns 1, 2, 3 and exon

5. Intron 1 of the Oct4 gene in chicken, mouse and human sequences (Sprowles, Roelf and Cell Biology 410, unpublished data) (Data not shown).

Epigenetic regulation is another mechanism of Oct4 gene regulation. One of the best understood epigenetic mechanisms is DNA methylation, which involves the addition of a methyl group to CpG Island in the DNA strand. The DNA methylation of Oct4 promoter showed more un-methylated CpG islands in embryonic stem cells, indicating the activation of genes required for pluripotency. Studies also revealed that stem cells directed down the differentiation pathway exhibit methylation of the Oct4 promoter and thus repression of Oct4 expression (Hattori et al. 2004). Bisulfate sequencing of the Oct4 promoter showed less CpG methylated Islands on the Oct4 promoter in ESs compare with normal somatic cells (Medvedev et al. 2012). Nucleosome occupancy also plays a role in regulating the Oct4 expression; a nucleosome-positioning analysis technique

(NOMe-seq) was performed at the distal enhancer of Oct4; it showed nucleosome depletion during the pluripotent state and increased nucleosome occupancy at the distal

8 enhancer regulatory regions of Oct4 when cells received the differentiation signal (You et

al. 2011).

The human Oct4 gene can encode at least three transcripts by alternative splicing:

Oct4A-Oct4B-and Oct4B1 (Atlasi et al. 2008). These isoforms share identical POU

DNA binding sites and C- terminal transactivation domains but differ in their N termini.

Oct4A has been showed to be located in the nucleus and expressed in human ES/EC

cells. It defined by a unique exon 1 and has been considered to be responsible for

pluripotency marker (Lee et al. 2006; Atlasi et al. 2008). The isoform Oct4B is thought to

be expressed in somatic cells and to be located in the cytoplasm of non-pluripotent cells.

Oct4B1 is expressed in human ES and EC cells and it may have a potential role in

maintaining the pluripotent state of human ES and EC. Oct4B1 has a novel exon 2b that

gives it a unique feature that helps scientists to distinguish between Oct4B1 and Oct4B

(Atlasi et al. 2008). To date the Oct4B and Oct4B1 isoforms have not been described in

mESCs.

Figure 2. The identification of AP-1 sites in the regulatory sequences of Mus musculus (mouse). Bioinformatics analysis identified AP-1 binding sites in the Oct4 gene at the promoter and the first intron of mouse.

9 The Transcription Factor cJun

The transcription factor cJun functions as a homodimer or heterodimer to function

as the transcription factor activator protein- 1 (AP-1), which is comprised of dimers

formed by proteins of the Jun (cJun, JunB, JunD) and Fos (c-Fos, Fos B, Fra1, and Fra2)

families. cJun had been shown to be autoregulated through its product in a positive

regulation loop (Angel et al. 1988). Its transcriptional activity is regulated in part by its

upstream regulatory kinase, Jun N Terminal Kinase (JNK). JNKs are activated by JNK

kinases including MKK4 and MKK7 (Estus et al. 1994; Ham et al. 1995; Virdee et al.

1997; Eilers et al. 1998; Davis 2000). Full transcriptional activity of cJun requires its phosphorylation at serine 63 and 73 by JNK. For this to occur, JNK must physically associate with cJun on leucine 40/42 (Sprowles and Wisdom 2003; Weiss and Bohmann,

2004).

Biological Roles of the Transcription Factor cJun and JNK

Both cJun and JNK play significant roles in cellular activities such as

proliferation, apoptosis, differentiation, mRNA splicing and tissue morphogenesis

(reviewed in Meng and Xia, 2011). An immediate early gene, transcriptionally active

cJun regulates the progression of the G1 phase in the cell cycle, which controls diverse

cellular processes such as proliferation, cell cycle arrest allowing for DNA repair,

terminal differentiation, and apoptosis (Hunter and Pines, 1994; Weinberg et al.

1995). Murine embryonic fibroblasts deficient in cJun show significantly altered growth

properties due to a defective cell cycle progression through its direct regulation of cyclin

10 D1 expression (Johnson et al. 1993; Schreiber et al. 1999; Wisdom et al. 1999). The

cJun/JNK pathway also regulates apoptosis. Murine embryonic fibroblasts lacking cJun

exhibit growth arrest after prolonged UV exposure (Shaulian et al. 2000). cJun also

inhibits the expression of the tumor suppressor p53, and therefore its downstream target

p21 (Schreiber et al. 1999). Reviewed in Shaulian and Karin 2002, cJun directly regulates

the transcription of cell survival and apoptosis genes, including FasL, Bim, Bcl3 and Fas.

The balance between the pro- and anti- apoptotic genes determines the fate of the cells to

survive or to go under apoptosis. JNK regulates the cell cycle under normal growth

conditions by increasing the ubiquitin-dependent degradation of c-Myc (Vargas and

Ronai, 2004). JNK also play roles in cell survival and apoptosis; prolong inhibition of

JNK resulted in differentiation, apoptosis and inhibition of cell proliferation within the central nervous system (Yang et al. 2005).

The JNK/cJun pathway has also been shown to play significant roles in embryonic development. Inactivation of cJun in mice results in embryonic lethality between day 12.5 and 13.5 from defects in the formation of normal cardiac outflow tract and abnormalities in the formation of liver (Eferl et al.1999; Schorpp-Kistner et al. 1999;

Hilberg et al. 1993). Mouse fetuses that lack JNK1 and JNK2 expression died at

embryonic day 11 due to defective neural tube formation (Sabapathy et al. 1999). In the

developmental stages, cJun is required in hepatocyte proliferation during liver

development and regeneration. cJun null mice had decreased hepatocyte proliferation,

and post partial hepatectomy, the liver regeneration was impaired and 50% of the cJun

mutant cells died (Behrens et al. 2002). Another study showed the interaction of multiple

11 signaling pathways including AP-1 (cJun and c-Fos), p38, extracellular signal-regulated kinase (ERK) 1, 2, and nuclear factor-κB (NF- κB) with the small molecule icariin lead to cardiomyogenic cell lineage differentiation of mouse ES cells (Wo et al. 2008). JNK has been found to be a negative regulator of BMP2, of which expression is essential in osteoblastic differentiation through the activation of MAPK cascade signals that activate

Runx2 expression and result in initiating osteoblastic differentiation process in murine multipotent mesenchymal progenitor cell and preosteoblastic cell line.

A study showed the inhibition of JNK resulted in increasing BMP2-induced osteoblastic differentiation in multipotent C2C12 and preosteoblastic MC3T3- E1 cell lines (Huang et al. 2012). JNK signaling pathway has been shown to play a significant role in neuronal development. Oliva et al 2006 showed the phosphorylated form of JNK to be required for axonogenesis. The authors showed the inhibition of JNK by the addition of SP600125, a specific inhibitor of JNK, resulted in inhibiting axonogenesis.

However, it did not prevent their differentiation into dendrites (Oliva et al. 2006). JNK also regulates significant neurons function such as memory formation, neuronal death, and brain morphogenesis (Reviewed in Coffey 2004).

Limited roles for cJun/JNK signaling has been observed in embryonic stem cell biology. JNK deficient mESCs exhibited rapid and high proliferation and several defects in lineage specific differentiation compare to the wild type, suggesting the importance of

JNK in the differentiation and proliferation of embryonic stem cells but demonstrate that it is not required for the self-renewal (Xu and Davis , 2010). To date we have not been

12 able to find significant evidence of a role for cJun in the inner cell mass or embryonic

stem cell potency.

cJun was discovered as the tumor promoting factor in Avian Sarcoma Virus 17

(Cavalieri et al 1985). Since then, both cJun and JNK also have been shown to play roles

in cellular transformation and tumor formation. Apostolou et al 2013 preformed a study

in human colon cancer stem cells that indicated a possible relationship between AP-1 and the maintenance of cancer stem cells. The data from the study showed the knockdown of cJun resulted in 130% increase of Oct4 in human colon cancer stem cells (Apostolou et al. 2013). Another study suggested Lamine-1 to highly induce the activation of phospho- cJun (S63) through the JNK PI3K/Akt and ERK pathways in human bone marrow mesenchymal stem cells (MSCs). The data showed the phosphorylation of cJun S63 resulted in the activation of α6 integrin and neurofilament-L genes that results in neurite outgrowth in human MSCs (Mruthyunjaya et al. 2011). Moreover, a study showed cJun and β-catenin/TCF4 positively regulated Nanog in human colorectal cancers (Ibrahim et al. 2012), another study showed Nanog regulates the expression of cJun through the binding of its promoter in cancer cells (Lin et al. 2014). Preliminary studies in our research group identified changes in the expression of Oct4 in Chicken fibroblast cells expressing a mutant form of cJun (L40/42A) and TCF4 (dnTCF4) (data not shown)

Taken together, the implications and roles of cJun and JNK had been investigated to some degree; however, the roles of cJun and JNK in the early embryonic development, embryonic stem cells and implications in cancers needs further understanding.

13 Specific Aims:

Oct4 is known as a key regulator of pluripotency in embryonic stem cells.

Aberrant expression of Oct4 has been reported in a number of cancers (Uche et al.

2005; Weiren et al. 2013). Therefore, investigating the regulation of the Oct4 gene is an

important step toward understanding the pluripotency state, differentiation and cellular

transformation. The transcription factor cJun and its upstream activator JNK play roles in

cell cycle, proliferation, embryonic development, differentiation and cancer (Jochum et

al. 2001; Amura et al. 2005; Apostolou et al. 2013). However, the literature has limited

support for a direct regulation of Oct4 by the transcription factor cJun and its upstream

activator. Our laboratory identified a putative AP-1 binding site approximately 2500 bp upstream of the putative Oct4A transcription start site in two different species. Thus, this study investigated the role of JNK/cJun on the regulation of Oct4 expression and stem cells potency in mESCs. This study gives insights in understanding pluripotency, early embryonic differentiation and cellular transformation by addressing the following specific aims:

1. Determining the role of cJun activity on Oct4 expression in murine embryonic stem

cells.

We addressed this question by modulating cJun activity in murine embryonic

stem cells and observing changes in Oct4 expression. Murine embryonic stem cells were

either cultured the presence of drugs that modulated JNK activity or were transfected

with plasmids that overexpress GFP cJun or GFP cJun mutants with reduced or increased

14 transcriptional activity (Sprowles and Wisdom 2003). Changes in the Oct4 expression was analyzed through immunocytochemistry and Western blot analysis.

2. Evaluating the role of cJun signaling on mESC potency.

This objective was addressed by testing the ability of mESCs transiently transfected with GFP cJun and GFP cJun mutants to remain pluripotent and differentiate in vitro. Transfected cells were assayed for alkaline phosphatase expression and directed differentiation to insulin secreting Pancreatic-islet-like clusters.

3. Evaluating the role of JNK signaling on mESC potency.

We addressed this question by testing the ability of mESCs with modulated JNK signaling to remain pluripotent and differentiate in vitro. Murine embryonic stem cells were cultured in the presences of drugs that affect JNK activity. The cells were tested for changes in alkaline phosphatase expression and their ability to differentiate to insulin secreting Pancreatic-islet-like clusters and beating cardiomyocytes.

15 MATERIALS AND METHODS

293T Cell Culture

Lenti-XTM 293T cells (Clontech, Mountain view, CA) were cultured in 90%

Dulbecco's Modified Eagle Medium (DMEM, Life Technologies, Grand Island, NY) and

10% fetal bovine serum (Life Technologies, Grand Island, NY) or 10% fetal Bovine

Serum for Cell Culture (Tetracycline-Free) (Clontech, Mountain View, CA) and were

maintained at 37°C in 5% CO2. 293T cells were thawed from LN2 storage in a water bath

at 37ºC for 1-2 minutes. Thawed cells were transferred into a sterile 15 ml conical tube

with 5 ml of pre-warmed media. The cells were centrifuged at 120 x g for 5 minutes and the supernatant was discarded. The cells were resuspended in 2 ml of fresh media. Cell number and viability were assessed by Trypan Blue analysis, in a 1:10 dilution with a

0.4% Trypan Blue solution (Life Technologies, Grand Island, NY). The solution was incubated for 5 minutes; 10 µl of the mixture was loaded on both sides of a hemocytometer and the number of cells counted by eye. Cells were plated at 30,000-

50,000 cells/cm2. Media was changed when the media color was orange or yellow,

indicating proton accumulation. The cells were sub-cultured when they reached 80-90%

confluence by removing old media, washing with sterile phosphate-buffered saline

without Mg2+ or Ca2+ (PBS, Life Technologies, Grand Island, NY) and adding 0.05%

trypsin-EDTA (Life Technologies, Grand Island, NY) for 5 minutes. Cells were then

transferred into a sterile 15 ml conical tube containing 10% FBS in DMEM, centrifuged

16 at 120 x g for 5 minutes, and resuspended in 2 ml fresh media. Cells number and viability

was assessed by the assay and plated at 30,000-50,000 cells/cm2.

J1 and B6/BLU Murine Embryonic Stem Cell Culture

Two different mouse embryonic cell (mESCs) lines were used in this study: the J1

cell line derived from a 129S4/SvJae male blastocyst (ATCC Cat# SCRC1010) and

B6/BLU line derived from a C57BL/6Tac male blastocyst (ATCC at# SRC1019)

(American Type Culture Collection (ATCC), Manassas, VA). Both cell lines were

cultured under the following identical conditions. Mouse embryonic stem cells were

plated on a feeder layer of 30,000-50,000 cell/cm2 of irradiated or mitomycin C treated p2 mouse embryonic fibroblasts (MEFs, CytoSpring LLC, Mountain View, CA) at

30,000-50,000 cells/cm2 in culture dishes pre-treated with 0.1% gelatin solution

(STEMCELL Technologies Inc., Vancouver, BC, Canada or EMD Millipore, Temecula,

CA). Cells were maintained at 37°C in 5% CO2 in mESC culture media (1% of 2.0 mM

L-alanyl-L-Glutamine (STEMCELL Technologies Inc., Vancouver, BC, Canada), 1% of

1x non-essential amino acids (STEMCELL Technologies Inc., Vancouver, BC, Canada),

0.1% of 1000x 2-mercaptoethanol (Sigma- Aldehyde®, Louis, MO ), 0.1% of ESGRO®

Mouse Leukemia Inhibitory Factor (LIF) (EMD Millipore, Temecula, CA), 15% of fetal

bovine serum and 82.8% of DMEM (Life Technologies Life Technologies, Grand Island,

NY and ATCC, Manassas, VA ). MESCs were sub-cultured at 40-50% confluence (for

detailed methods see “Cell culture of 293T”).

17 Chemical Treatment of Mouse Embryonic Stem Cells and 293T

Mouse embryonic stem cells and 293T were treated with either 50 ng/ml

anisomycin (EMD Millipore, Temecula, CA), 10 ng/ml anisomycin, 50 µM JNK

inhibitor SP600125 (Cell signaling Technology Inc., Danvers, MA), or 10 µM JNK

inhibitor SP600125 for the time period specified/each experiment. Both anisomycin and

JNK inhibitor SP600125 were reconstituted in sterile dimethyl sulfoxide (DMSO, Sigma-

Aldehyde®, Louis, MO), and a negative control of DMSO was used.

Protein Extraction and Western Blot

Cells were harvested for protein by first discarding old media and washing with

sterile phosphate-buffered saline without Mg2+or Ca2+ (PBS, Life Technologies, Grand

Island, NY). The cells were dissociated with Trypsin-EDTA (Life Technologies, Grand

Island, NY) for 5 minutes and the cell suspension was transferred into a 15 ml conical

tube with 4 ml media then centrifuged at 120 x g for 5 minutes. Cells were counted using

the Trypan Blue assay (described above). One million cells were transferred into a sterile

1.5 ml microfuge tube and centrifuged at 120 x g for 1-2 minutes. The supernatant was discarded and the cell pellet washed with 200 µl of sterile PBS w/o Mg2+ or Ca2+ and centrifuging at 120 x g for 1-2 minutes. The supernatant was discarded and the cell pellets were harvested for protein or saved at -80ºC for future protein extraction.

Nuclear Cytoplasmic fractionation was performed using NE-PER™ Nuclear and

Cytoplasmic Extraction Reagents (Thermo scientific, Rockford, IL) following the

manufacturer’s instructions. 100 µl of ice-cold CRE I was added to each pellet of one

18 million cells and the solution was vortexed for fifteen seconds, followed by a ten minute

incubation on ice. The addition of 5.5 µl ice-cold CRE II was applied and the mixture was vortexed for five seconds and incubated on ice for one minute. The mixture was vortexed for five seconds and centrifuged at 13,000 x g for five minutes. The supernatant containing the cytoplasmic extract was transferred into a pre-chilled 1.5 ml sterile tube and incubated on ice with 1x Halt™ Phosphatase Inhibitor Cocktail (Thermo Scientific,

Waltham, MA). The pellet containing the nuclear extract was suspended in 50 µl of ice- cold NER and vortexed for 15 seconds. The mixture was incubated on ice for 40 minutes while vortexing every 10 minutes. The mixture was then centrifuged at 13,000 x g for 10 minutes and the supernatant was transferred to a pre-chilled 1.5 ml sterile tube with 1x

Halt™ Phosphatase Inhibitor Cocktail. 2 µl of the cytoplasmic and nuclear extracts were transferred to 1.5 ml sterile tubes to measure protein concentration and the rest of the extracts were stored at - 80 ºC.

Protein concentrations were determined using the Pierce™ BCA Protein Assay

Kit (Thermo Scientific, Rockford, IL) following the manufacturer’s directions. The BCA working reagents were prepared by mixing BCA reagent A with BCA reagent B at a ratio of (50:1, A:B). The working reagent was added at 50 µl to the 2 µl of extracted protein for the cytoplasm and nuclear extracts along with five diluted albumin (BSA) Standards

(provided). Mixtures were incubated at 37ºC for 30 minutes. The mixtures were then cooled to room temperature and the protein concentrations were determined using the

Nanodrop (Fisher Scientific, San Diego, CA). A standard curve was generated with the

19 BSA standard samples by the Nanodrop (Fisher Scientific, San Diego, CA) and this curve

was used to determine the concentration of the extracted proteins.

Whole cell extracts were generated by the addition of 50 µl of whole cell extract

buffer (4% 0.5M HEPES pH 7.7, 1% 5M NaCl, 0.2% 0.5 M EDTA, 0.05% Triton 100

and 94.8% ddH20) and 1x Halt™ Phosphatase Inhibitor Cocktail into a pellet of million

cells. Cells were vortexed and incubated for 15 minutes on ice followed by centrifuging

at 20,000 x g for five minutes. The supernatant was transferred into a 1.5 ml microfuge and the protein concentrations were determined using the Nanodrop (Fisher Scientific,

San Diego, CA).

Protein concentrations were normalized between samples and denatured in 1x

SDS sample buffer at 95 ºC for 5 minutes. (6x Sample buffer recipe listed in (Appendix II

Table 1). Five to six µg of protein was loaded into 10-20% gradient precast Novex Tris-

Glycine Protein Gels (Life Technologies, Grand Island, NY) along with 10 l of

PageRuler™ Plus Prestained Protein Ladder (Thermo Scientific, Waltham, MA). Gels

were run at 95 volts for three and a half hours in the presence of 1x running buffer at 4ºC

(see Appendix II for all buffer components Table 1). After running the gel, the gel

cassette was separated with a knife and the gel was transferred to a methanol soaked

Immobilon®-P PVDF membrane (EMD Millipore, Bellerica, MA). To prepare the

transfer, a tray was filled with transfer buffer, 4 blotting pads, and 2 pieces of filter

papers. The Immobilon®-PDVF transfer membrane was soaked in methanol for several

minutes. A transfer sandwich was then made in the tray consisting of 2 blotting pads

followed by a filter paper, the gel, the PVDF membrane, a filter paper and 2 blotting

20 pads. The blot module was secured and placed into the transfer chamber. The inner part

of the chamber was filled with running buffer (Appendix II Table 1) and the outer part

was filled with deionized water. The transfer was run at 25 volts for 2 hours. At the end

of the transfer, the PVDF membrane was incubated in blocking buffer (Appendix II Table

1) for two hours at 4ºC. Primary antibodies were incubated with the mESCs or 293T cells

at specific times and concentrations/antibody (Appendix II Table 2). The membranes

were then washed three times with 1x TBST and incubated for 45 minutes with the

appropriate secondary antibody at the appropriate conditions (Appendix II Table 2),

followed by three washes of 1x TBST for 15 minutes each. The membrane was soaked in

2 ml of WesternSure® PREMIUM Chemiluminescent Substrate (LI-COR, Lincoln, NA)

for 5 minutes and imaged by C-DiGit® Blot Scanner ( LI-COR, Lincoln, NA).

Immunocytochemistry

Cells were plated on coverslips pre-treated with either 2 µg/ml fibronectin

(STEMCELL Technologies Inc., Vancouver, BC, Canada) for 293T or murine embryonic fibroblast culture or 15 µg/ml of poly-L ornithine (Sigma- Aldehyde®, Louis, MO) for the pancreatic islet-like cluster differentiation protocol. Coverslips were incubated for one hour at 37 ºC and washed with PBS. 293T or MEF cells were plated onto coverslips and incubated for 24 hours at 37 ºC in 5% J1 or B6/BLU. Murine Embryonic Stem Cells were then plated onto MEF that covered the coverslips and incubated for another 24 hours.

21 Cells were fixed with 4% paraformaldehyde in PBS for 5 minutes.

Paraformaldehyde was discarded and cells were washed with PBS five times for 2

minutes each. The cells were then incubated with 0.1% Nonidet™ P 40 Substitute

(Sigma- Aldehyde®, Louis, MO) diluted in 1x PBS for 30 minutes. Cells were washed

twice with PBS for 2 minutes and incubated for 30 minutes in blocking buffer that

consisted of 0.1% Triton™ X-100 (Sigma- Aldehyde®, Louis, MO) and 1% goat serum

(Thermo fisher scientific, Waltham, MA) in PBS. After blocking, cells were incubated with primary antibody (Appendix II Table 3) for one hour followed by three washes with

PBS for 5 minutes each. Secondary antibody (Appendix II Table 3) was added for one hour followed by three washes with PBS for five minutes each. Antibodies were diluted in blocking buffer (Appendix II Table 1). Coverslips with the stained cells were mounted on slides with 20 µl of ProLong® Gold antifade reagents with DAPI (Life Technologies,

Grand Island, NY). Cells were analyzed using Zeiss microscope and software (Carl Zeiss

Inc., Thornwood, NY) using 350 nm excitation and 450 nm emission filter to visualize

DAPI, 595 nm excitation and 615 nm emission filters to visualize Texas Red and 490 nm excitation and 520nm emission filter was used for the visualization of green fluorescence.

Transient Transfection of 293T Cells

Transfection of 293T cells was performed according to Lipofectamine™

2000 (Life Technologies, Grand Island, NY) manufacturer instructions. 293T cells were plated into a 6 well tissue culture plates and transfected with a wild type GFP cJun clone, mutant GFP cJun L40/42A, or mutant GFP cJun R54A. The transfection of pLVX-EF1α-

22 AcGFP1-C1 empty vector was performed as a negative control. Each transfection

reaction included two tubes; one tube containing 1.6 mg of the cJun clone and 100 µl of

Media 199 (Life Technologies, Grand Island, NY), and the other tube containing 100 µl

of Media 199 and 4μl of Lipofectamine ™ 2000. Each tube was incubated for 5 minutes,

and then the contents of the tubes were combined. The reaction was incubated for 20

minutes at room temperature, then transferred to the plated 293T cells. Transfected 293T

cells were incubated for 48 hours at 37 ºC prior to harvesting for protein or fixation for

immunocytochemistry.

Transient Transfection of Mouse Embryonic Stem Cells

Twenty four well tissue culture plates were pre-treated with 0.1% gelatin for 30

minutes. Mouse embryonic stem cells were then plated and the cells were incubated in at

37ºC and 5% CO2 for 5 hours prior to transfection. Cells were transfected with pLVX-

EF1α-AcGFP1 plasmids containing either GFP cJun, GFP cJun L40/42A, GFP cJun

R54A, or no insert (GFP alone) (See Appendix 1 for construct details). Each transfection

consisted of two tubes, one tube contained 75 µl of Opti-MEM® I Reduced Serum

Medium (Life Technologies, Grand Island, NY) and 9 µl of Lipofectamine® 3000

Transfection Reagent (Life Technologies, Grand Island, NY). The second tube contained

75 µl of Opti-MEM® I Reduced Serum Medium, 1.3 µg of each EF1α-AcGFP1 plasmid, and 9 µl of P3000™ Reagent. Both tubes were mixed well and the contents of the tubes were combined. The mixture was incubated for 5 minutes then added to the mESCs.

Transfected cells were incubated for 6 hours and then harvested and plated into MEFs in

23 24 wells tissue culture plate. Transfected cells were incubated for 36 hours, and then fixed with 4% paraformaldehyde for immunocytochemistry.

Differentiation of Mouse Embryonic Stem Cells

Embyoid Body formation

Mouse embryonic stem cells were harvested and plated at 5x cells/cm2 in a 6-well ultra low adherent dish (Sigma- Aldehyde®, Louis, MO) in 3 ml of differentiation media

(15% of ES-Cult ™ fetal bovine serum (STEMCELL Technologies Inc., Vancouver, BC,

Canada), 10 mM of MEM Non- Essential Amino acids, 2 mM of L-Glutamine (Life

Technologies, Grand Island, NY), 1 mM of 1-Thioglycerol (Sigma- Aldehyde®, Louis,

MO ) and DMEM media (Life Technologies Life Technologies, Grand Island, NY and

American Type Culture Collection, Manassas, VA ). If the mESCs had been treated with aniosmycin or SP600125, the drugs were included at the appropriate concentration. Cells were incubated for two days. At day two, media was changed by transferring the media and embryoid bodies from each well to a sterile 15 ml conical tube. The Embryoid bodies were allowed to settle in the bottom of the conical tube for 5 minutes. Old media was removed and 3 ml of new differentiation media was added. EBs were plated back into the same ultra low adherent wells and incubated for another 48 hours. At day 4 from the start of plating EBs in low adherent dish, EBs were differentiated into either cardiomyocytes or pancreatic islet cells.

24 Directed differentiation from EBs to beating cardiomyocytes

Formation of cardiomyocytes was performed by harvesting EBs at day four and

transferring them onto 0.1% gelatin coated tissue culture plate in the presence of the same

differentiation media used for embryoid body formation. Media was changed every 48

hours and cultures were monitored for autonomously beating cell clumps.

Directed differentiation from EBs to Pancreatic islet-like insulin secreting clusters

Formation of pancreatic islet-like clusters was performed following the protocol

published in the technical manual from StemCell Technologies entitled “In Vitro

Differentiation of Murine ES Cells into Pancreatic Islet-like Clusters, Version 1.0.1,

February 2003” (STEMCELL Technologies Inc., Vancouver, BC, Canada). EBs were

harvested at day 4 and transferred into tissue culture plates in the presence of serum-free

ITS-A medium (1x of ITS supplement-A 100x (STEMCELL Technologies Inc.,

Vancouver, BC, Canada), ES-Cult ™ Basal medium-A (STEMCELL Technologies Inc.,

Vancouver, BC, Canada). Cells were cultured for 6 days with media changes every 48

hours to enrich for nestin+ cells. At day 6, the expansion of pancreatic precursor cells

was performed. Cells were harvested and transferred into a 15 ml conical tube and cell

clumps were allowed to settle for 5 minutes. The supernatant were transferred into a new

15 ml conical tube without transferring any of the clumps. The supernatant was

centrifuged at 172 x g for 5 minutes followed by discarding the old media and re-

suspending the cells in pancreatic proliferation media (ES-Cult ™ Basal medium-A

(STEMCELL Technologies Inc., Vancouver, BC, Canada) with 1x of N2 supplement-A

100x (STEMCELL Technologies Inc., Vancouver, BC, Canada), 1x of B27 supplements

25 50x (Life Technologies, Grand Island, NY), 25 ng/ml of FGF-b recombinant human (Life

Technologies, Grand Island, NY). Cells were plated on 15 µg/ml of poly-L-ornathine treated coverslips at 2x106 cells in a 12-wells plate. Cells were incubated in pancreatic proliferation media for 6 days and media was changed every 48 hours. At day 6, the media was changed to pancreatic differentiation medium (ES-Cult ™ Basal medium-A with 1x of N2 supplement-A 100x, 1x of B27 supplements 50x, 10 mM of nicotinamide

(STEMCELL Technologies Inc., Vancouver, BC, Canada) to induce insulin-secreting pancreatic islet-like cluster formation. Cells were incubated for 6 days and media was changed every 48 hours. At day 6, cells were fixed for immunocytochemistry to test for enrichment of pancreatic islet cell markers.

Alkaline phosphatase

MESCs at 40% confluence were fixed with 4% paraformaldehyde for two minutes followed by rinsing with 1x TBST (Appendix II Table 1) and treated with the alkaline phosphatase (AP) detection kit (EMD Millipore, Temecula, CA). Treated cells were incubated in dark at room temperature for 15 minutes. AP reagents were aspirated and another rinsing step with 1x TBST was performed. Cells were covered with 1x PBS and the counting of colonies expressing AP was performed under light microscope.

26 Statistical Analysis

Statistical tests were performed using SigmaPlot 12 software (Systat Software).

Parametric ANOVAs were used to analyze the data. The data was described as the mean

± the SEM.

RESULTS

The Role of the JNK/cJun Pathway on Oct4 Expression in Murine Embryonic Stem Cells

Our first aim explores the hypothesis that the JNK/cJun pathway regulates Oct4 gene expression. This could be a consequence of modified transcription factor binding, changes in splicing events to result in variant isoform expression, or a combination of both. To address this question, murine embryonic stem cells (mESCs) were treated with drugs that affect JNK activity or transfected with plasmids that overexpress GFP, GFP cJun, or GFP cJun mutants with either reduced or increased transcriptional activity. Oct4 expression was analyzed by immunocytochemistry and western blot.

Examining the modulation of JNK/cJun pathway by chemical treatment in 293T

JNK activity was stimulated by anisomycin, a potent chemical activator of JNK, and inhibited with the JNK pharmacological inhibitor SP600125 (Cano et al 1996,

Hazzalin et al 1998, Bennet et al. 2001). We began by using these chemicals in 293T cells to confirm the drug treatments were working as anticipated. Anisomycin simulation of the JNK/cJun pathway can be confirmed by increased JNK phosphorylation on the activating Thr X-Tyr motif and cJun phosphorylation on S63 and S73. Treatment of cells with SP600125 results in reduction of cJun phosphorylation at these same residues, but does not reduce JNK phosphorylation below background as the inhibitor associates with the enzyme’s catalytic domain (Bennet et al 2001).

28 Immunocytochemistry data from 293T cells treated with Anisomycin at both 50 ng/ml

and 10 ng/ml for 30 minutes and incubated with cJun antibody resulted in a higher cJun

signal (Abcam #32137), suggesting increased levels of endogenous protein. 293T cells

treated with 50 μM SP600125 and 10 μM SP600125 for 30 minutes showed decreased

cJun expression (Figure 3). Since cJun is autoregulatory, the changes in total cJun protein

confirmed successful modulation of the system using the chemical treatments.

The amount of phosphorylated cJun under these conditions was also

characterized. Antibodies to both phosphorylated cJun S63 (p- cJun S63, Abcam #32385)

and phosphorylated cJun S73 (p-cJun 73, Cell signaling # 3270s) showed increased

signal in response to anisomycin treatment that seemed to be enriched in nucleus (Figure

4,5). 293T cells that were treated with 50 μM SP600125 showed cytoplasmic

localization of p-cJun S63 (Figure 4). No phosphorylated cJun S73 was observed in cells treated with the inhibitor.

Immunocytochemistry in 293T cells using a JNK antibody (Calbiochem #

559304) showed nuclear and cytoplasm signal of JNK in treated samples and the control

(Figure 6A). Immunocytochemistry with an antibody to the phosphorylated form of JNK

(p-JNK antibody, Abcam # ab124956) showed a nuclear and cytoplasm signal for all samples (Figure 6B).

Figure 3. Chemical modulation of the JNK pathway affects the levels and subcellular localization of cJun in 293T cells. 293T cells treated for 30 minutes with either SP600125 or anisomycin showed increased expression of cJun (n=2). The signal is predominantly nuclear. Images were taken at 20X. Scale bar = 20 μM.

Figure 4. Modulating the JNK pathway by chemical treatment affects the levels and the subcellular localization of p-cJun 63 in 293T cells.. An increase in p-cJun S63 was observed in 293T cells after treatment with Anisomycin at 50 ng/ml and 10 ng/ml, which showed higher expression of p-cJun S63. The signal seems predominantly localized to the nucleus. Cytoplasmic signal was observed in samples treated with SP600125 at 50 μM. Both nuclear and cytoplasmic signal was observed in samples treated with 10 μM SP600125, which also showed lower p-cJun S63 expression. Scale bar = 20 μM. Arrows= cytoplasmic localization.

Figure 5. Modulating the JNK pathway by chemical treatment affects the levels of p-cJun S73 in 293T cells. Increased signal of p-cJun S73 was observed in 293T cells that were treated with 50 ng/ml and 10 ng/ml. The signal seems to be enriched in the nucleus. (n=2) for all experiments. Each chemical treatment was performed for 30 minutes. Images were taken at 20X. Scale bar = 20 μM.

Figure 6. p-JNK expression levels in 293T cells treated with anisomycin or SP600125. A) Nuclear signal of JNK among treated samples. B) Treatment with anisomycin at 50 ng/ml and SP600125 at 50 μM showed similar p-JNK expression. Treatment with anisomycin at 10 ng/ml and SP600125 at 10 μM showed also showed similar p-JNK expression level. n=2 for all experiments. Images were taken at 20X. Each chemical treatment was performed for 30 minutes. Scale bar = 20 μM. 33 To directly address how JNK stimulation affected protein expression and

phosphorylation, 293T cells were treated with anisomycin and SP600125 for two hours

followed by Western blot analysis. Cells treated with anisomycin had higher levels of

phosphorylated cJun S63 and S73 when compared to cells treated with SP600125 and

DMSO. Cells treated with anisomycin at 50 ng/ml had almost complete loss of p-cJun

S63 and S73 from the nucleus accompanied by a significant increase of cytoplasmic signal (Figure 7).

Figure 7. Modulating JNK pathway by chemical treatments affects levels of p-cJun in 293T cells.Treatment with 10 ng/ml anisomycin resulted in increasing p-cJun S63 and S73 expression in the nuclear extraction. Treatment with 50 ng/ml anisomycin resulted in increasing p-cJun S63 and S73 expression in the cytoplasm extraction. Treatment was performed for two hours. N=1.

35 Examining the modulation of JNK/cJun pathway by chemical treatment on Oct4

expression in mESCs

Having characterized the effects of anisomycin and SP600126 on cJun and JNK

in 293T cells, we were interested in analyzing their effects on JNK/cJun signaling in

murine embryonic stem cells (mESCs). To identify the optimal chemical concentration,

J1 mESCs were treated with 50 ng/ml anisomycin, 10 ng/ml anisomycin, 50 µM

SP600125, 10 µM SP60012, or DMSO for 48 hours. The cells were then harvested and

tested for cell number and viability by Trypan Blue assay. The results showed the

addition of anisomycin at 10 ng/ml or 10 µM SP60012 did not affect the cells viability, but increasing the concentrations of anisomycin did. (Figure 8 and Table 1). Furthermore, the results showed significant decrease in cell number in samples treated with anisomycin at 10 ng/ml in comparison to samples treated with SP600125 at 10 μM and DMSO.

Increasing the drug concentration to 15 ng/ml and 50 ng/ml of anisomycin or 15 μM and

50 μM led to decrease the total cells number. The 15 ng/ml and 50 ng/ml of anisomycin

showed a higher significant decreasing of the cells number that were indicated by one

way ANOVA (p=0.004; F= 16.1; d.f.= 6) (p=0.02; F= 10.15; d.f.= 6) (Figure 8). mESCs,

cells were treated with 10 μM SP600125 in the presence or absence of LIF for six days

and tested for cell number and viability. Analysis by one-way RM ANOVA indicated a

significant decrease of cells number (p=0.001; F= 25.7; d.f. = 6) on samples with the

addition of SP600125 ± LIF (Figure 9).

Figure 8. Identification of the optimal concentration of anisomycin and SP600125 in J1 mESCs. Cells were treated with chemicals for 2 days and assayed for cell number and viability by Trypan Blue. Increasing the anisomycin and SP600125 concentration led to significant decrease in the cells number. This experiment was performed twice in triplicate.

Table 1. Treatment with SP600125 at 10 μM and anisomycin at 10 ng/ml did not change the viability of mESCs. MESCs were treated with chemicals for 2 days, cells were harvested and Trypan Blue assay was performed. Experiment was performed twice in triplicate.

Treatments Viability percentage SEM

Anisomycin at 10 ng/ml 100% ± 0 SP600125 at 10 μM 100% ± 0 DMSO 100% ± 0 Anisomycin at 50 ng/ml 70% ± 0.1 SP600125 at 50 μM 100% ± 0 DMSO 100% ± 0

Figure 9. SP600125 ± LIF reduces the number of J1 mouse embryonic stem cells. Cells were under treatment for 6 days. Post treatment cells were harvested and counted. This experiment was performed twice in triplicate. N=2.

39 Murine embryonic stem cells of both the J1 and B6/BLU cell lines were treated with either DMSO, anisomycin at 50 ng/ml, anisomycin at 10 ng/ml, SP600125 at 50 µM or SP600125 at 10 µM for two hours. Addition of anisomycin at 50 ng/ml resulted in

JNK expression in both cytoplasm and nucleus at a percentage of 63.3:36.7% respectively (Figure 10A and Table 2). The expression of JNK was observed in the cytoplasm of mESCs treated with anisomycin at 10 ng/ml and SP600125 at both 50 μM and 10 μM. The addition of p-JNK antibody did not show expression differences among samples (Figure 10B). However, Western blot analysis in B6/BLU cell line showed the treatment with Anisomycin at 10 ng/ml and 50 ng/ml for two hours resulted in an increase in the nuclear fraction for P-JNK. This Western blot was performed on one set of samples (Figure 11).

Figure 10. Anisomycin at 50 ng/ml resulted in increases the nuclear localization of JNK. A) mESCs treated with anisomycin at 50 ng/ml showed nuclear and cytoplasmic localization of JNK. The other treatments resulted in cytoplasmic localization. B) The addition of p-JNK antibody did not result in obvious changes of P-JNK expression level. N=2. Experiment was performed in J1 and B6/BLU cell lines; presented images were from the B6/BLU cell line. Treatment was performed for two hours. Images were taken at 63X.

Table 2. The addition of anisomycin at 50 ng/ml resulted in increased nuclear localization of JNK in mESCs. Data were collected from 80 mESC colonies from the B6/BLIU cell lines. Experiment was performed twice in duplicate for immunocytochemistry data.

Treatment % of JNK expression % of JNK expression in the cytoplasm of in the cytoplasm and mESCs nucleus of mESCs

Anisomycin at 50 ng/ml 63.3% 36.7%

Anisomycin at 10 ng/ml 100% 0%

SP600125 at 50 μM 100% 0%

SP600125 at 10 μM 100% 0%

DMSO 100% 0%

No treatment 100% 0%

Figure 11. Modulating JNK pathway by chemical treatments for 2 and 4 hours showed increase p-JNK in the nuclear fraction in mESCs. 1) 50 ng/ml anisomycin. 2) Anisomycin at 10 ng/ml. 3) 50 μM SP600125 4) SP600125 at 10 μM. 5) DMSO. 6) No treatment. Experiment was performed one time in each cell line in B6/BLU.

43 We next tested the effect of the chemical treatments on cJun and phosphorylated cJun. Murine embryonic stem cells were treated with DMSO, anisomycin 50 ng/ml, anisomycin 10 ng/ml, 50 µM SP600125, or 10 µM SP600125 for two hours. Both J1 and B6/BLU cells treated with anisomycin showed an overall increase in total p-cJun

S73 and p-cJun S63, with more accumulation in the nucleus when compared to the other samples (Figure12, 13; Table 2). Western blot analysis confirmed the immunocytochemistry data. At 4 hours, samples treated with anisomycin at 50 ng/ml and 10 ng/ml also showed an increase in the expression level of p-cJun S63 and p cJun

S73 in the nuclear fraction of B6/BLU cell line (Figure 14). This experiment was not performed in J1 cells.

Western blot analysis of B6 mESCs verified that cJun had a shift in subcellular localization at 2 and 4 hours. At 2 hours samples treated with Anisomycin at 50 ng/ml showed cJun, p-cJun S63 and p-cJun S73 in both cytoplasm and nuclear extraction, samples treated with Anisomycin at 10 ng/ml showed p-cJun S73 to be present in the nuclear extraction only and p-cJun S63 in the cytoplasm extraction only. The same sample showed cJun in both the cytoplasm and nuclear extraction. On the other hand, samples treated with SP600125 at 50 µM and 10 µM resulted in the absence of p-cJun

S63 in the nuclear extraction but it was present in the cytoplasmic extraction. The no treatment control sample showed p-cJun S73 in the nuclear extraction and p-cJun S63 was observed in the cytoplasmic extraction (Figure 14). At 4 hours post treatment, samples treated with anisomycin at 50 ng/ml and 10 ng/ml continued to showed significant p-cJun S73 in nuclear extraction but nuclear p-cJun S63 was hardly

44 detectable. It was found in the cytoplasmic fraction (Figure 14). Tables 4 and 5 have a summary of cJun subcellular localization patterns in both mESC cell lines and 293T cells. Note that although cJun and its transcriptionally active form exhibited subcellular localization differences among untreated 293T and untreated mESCs, the 293T cells were treated with chemicals for 30 minutes but the mESCs were treated for 2 hours.

(Figure 15).

Figure 12. Modulating JNK/cJun pathway by chemical treatments affects the subcellular localization of p-cJun S63 in mESCs. A) Cytoplasmic localization signal of p-cJun S63 was observed in mESCs post treatment with anisomycin at 10 ng/ml and SP600125 at both concentrations. Both cytoplasmic and nuclear signal was observed in samples treated with anisomycin at 50 ng/ml. B) The same images at panel A but at a higher magnification. Images were taken at 63X. Experiment was performed in J1 and B6/BLU cell lines, presented images were taken from the B6/BLU cell line. 46

Figure 13. . Modulating JNK/cJun pathway by chemical treatments showed nuclear subcellular localization of p-cJun S73 in mESCs. Nuclear localization signal of p-cJun S73 was observed in mESCs that were treated with drugs. A small increase in the p-cJun S73 after Anisomycin treatment was observed; n=2. Experiment was performed in two cell lines; presented images were taken for the B6/BLU cell line. Treatment was performed for two hours. Images were taken at 63

Table 3. The addition of anisomycin at 50 ng/ml resulted in cytoplasmic and nuclear localization of p-cJun S63. Data was collected from 76 mESC colonies from J1 and B6/BLU cell lines.

Treatment % of p-cJun S63 % of p-cJun S63 SEM expression in the expression in the cytoplasm cytoplasm and nuclear

Anisomycin at 50 ng/ml 65.4% 34.6% ± 1.22 Anisomycin at 10 ng/ml 100% 0% ± 0 SP600125 at 50 μM 100% 0% ± 0 SP600125 at 10 μM 100% 0% ± 0 DMSO 100% 0% ± 0 No treatment 100% 0% ± 0

Figure 14. . Modulating JNK pathway by chemical treatments for 2 and 4 hours affects the subcellular localization of p-cJun and the level of p-cJun in mESCs. A. B6/BLU cell lines: 1) Anisomycin at 50 ng/ml. 2) Anisomycin at 10 ng/ml. 3) SP600125 at 50 μM. 4) SP600125 at 10 μM. 5) DMSO. 6) No treatment. B. J1 cell lines at 2 hours: 1) Anisomycin at 50 ng/ml. 2) Anisomycin at 10 ng/ml. 3) SP600125 at 10 μM. 4) DMSO. C. Label for J1 cell lines at 4 hours: 1) DMSO. 2) Anisomycin at 10 ng/ml. 3) SP600125 at 50 μM. 4) SP600125 at 10 μM. Experiment was perfumed one time in each cell line (B6/BLU and J1).

Table 4. Comparison of the subcellular localization and expression of p-cJun S63 between 293T and mouse embryonic stem cells with the addition of SP600125 and anisomycin. The 293T cells were treated with chemicals for 30 minutes but the mESCs were treated for 2 hours.

Treatments Immunocytochem Immunocytochemistry Western Western of istry of p-cJun of p-cJun S63 in of p-cJun p-cJun S63 S63 in 293T mESCs S63 in in mESCs 293T

30 min. 2 hrs. 30 min. 2 hrs.

Anisomycin Nuclear expression Nuclear and Cytoplasm Cytoplasm Cytoplasm at 50 ng/ml expression expression expression Anisomycin Highest Nuclear Cytoplasm expression Nuclear Cytoplasm at 10 ng/ml expression expression expression SP600125 Cytoplasm Cytoplasm expression Nuclear Cytoplasm at 50 µM expression expression expression SP600125 Nuclear and Cytoplasm expression No Cytoplasm at 10 µM cytoplasm expression expression expression DMSO Nuclear expression Cytoplasm expression Nuclear Cytoplasm expression expression No Nuclear expression Cytoplasm expression Nuclear Cytoplasm treatment expression expression

Table 5. Comparison of the subcellular localization and expression of p-cJun S73 between 293T and mouse embryonic stem cells post the addition of SP600125 and anisomycin. The 293T cells were treated with chemicals for 30 minutes but the mESCs were treated for 2 hours.

Treatments Immunocytoch Immunocytoc Western Western emistry of p- hemistry of p- of p-cJun of p-cJun cJun S73 in cJun S73 in S73 in S73 in 293T mESCs 293T mESCs

30 min 2 hrs. 30 min 2 hrs.

Anisomycin Nuclear Higher Nuclear Cytoplasm Nuclear at 50 ng/ml expression expression expression expression Anisomycin Nuclear Nuclear Nuclear Nuclear at 10 ng/ml expression expression and expression cytoplasm expression SP600125 Cytoplasm Nuclear Nuclear Nuclear at 50 µM expression expression and expression cytoplasm expression SP600125 Nuclear and Nuclear Nuclear Nuclear at 10 µM cytoplasm expression expression expression expression DMSO Nuclear Nuclear Nuclear Nuclear expression expression expression expression No Nuclear Nuclear Nuclear Nuclear treatment expression expression expression expression

A B C

Figure 15. . Different subcellular localization of c-Jun and phospho c-Jun among untreated 293T and mESCs. A) C-Jun expression was observed in the cytoplasm and nucleus of mESCs but it was only in the nucleus of 293T cells. B) P-cJun at serine 63 showed cytoplasm signal in mESCs and it was nucleus in 293T cells. C) P-cJun at serine 73 showed nucleus signal in mESCs and 293T cells. N=3.

52 Having optimized the conditions for anisomycin and the SP600125 in mESCs and verified they had the appropriate effect on JNK/cJun signaling, we were interested in evaluating their effect on Oct4 expression. To do this we selected two different antibodies to Oct4—one that recognizes all characterized Oct4 isoforms by binding at

300 residues to the C-terminus of Oct4 (Abcam # ab19857) and another that only recognizes protein sequences of the Oct4A isoform (Cell signaling # 2840s).

Immunocytochemistry data was collected from two mESC lines including J1 and

B6/BLU. Our analysis did not show difference in total Oct4 or Nanog expression in response to the chemical treatments. However, there was a significant increase in the intensity level of Oct4A in samples treated with anisomycin at 50 and 10 ng/ml and 50

μM SP600125 (Figure 16). Data from Western blot analysis agreed with the immunocytochemistry data, showing an increase in the Oct4A protein level two hours post treatment with anisomycin at 50 and 10 ng/ml (Figure 17). Notably, the increase in the Oct4A expression agreed with the increase of p-cJun (see data above for details).

Figure 16. Oct4A expression is increased in mESCs treated with 50 and 10 ng/ml Anisomycin or 50 μM SP600125. A) Nuclear localization of Oct4 in mESCs among all samples with no differences in the Oct4 level.

B) Nuclear localization of Oct4A in mESCs among all samples with increase in the Oct4A level in samples treated with 50 and 10ng/ml Anisomycin or 50 μM SP600125.

C) Nuclear localization of Nanog in mESCs among all samples. Experiment was performed in two cell lines; presented images were taken for the B6/BLU cell line. Treatment was performed for two hours. Images were taken at 63X.

Figure 17. Modulating JNK pathway by chemical treatments for 2 and 4 hours affects the level of Oct4A in the nuclear fraction of mESCs. A. B6/BLU cell lines: 1) Anisomycin at 50 ng/ml. 2) Anisomycin at 10 ng/ml. 3) SP600125 at 50 μM. 4) SP600125 at 10 μM. 5) DMSO. 6) No treatment. B. J1 cell lines at 2 hours: 1) Anisomycin at 50 ng/ml. 2) Anisomycin at 10 ng/ml. 3) SP600125 at 10 μM. 4) DMSO. C. J1 cell lines at 4 hours: 1) DMSO. 2) Anisomycin at 10 ng/ml. 3) SP600125 at 50 μM. 4) SP600125 at 10 μM. Experiment was perfumed one time in each cell line (B6/BLU and J1).

55 Testing the Overexpression of cJun on Oct4

To test the role of cJun in the regulation of Oct4 expression, we compared Oct4 expression in murine embryonic stem cells expressing one of four different proteins: green fluorescent protein (GFP), GFP cJun, GFP cJun L40/42A, and GFP cJun R54A.

These cJun mutants were selected for specific properties. cJun L40/42A cannot bind

JNK. Therefore, it is not phosphorylated by the kinase and does not activate transcription through the classic AP-1 binding site as measured by luciferase assay. The mutant R54A shows slightly increased affinity for JNK, increased phosphorylation at cJun residues S63 and S73, and shows slightly increased transcriptional activity off consensus AP-1 binding sites (Sprowles and Wisdom 2003). If cJun regulates Oct4 through JNK activation/transcriptional activity, we expected to see changes in Oct4 expression that was consistent with the JNK regulatory properties of each of these cJun proteins.

Twice we attempted to generate stable mESC lines expressing the different forms of cJun. In the first round of the experiment, we used cJun constructs cloned into PLVX- pure lentiviral expression plasmids by previous students in the Sprowles lab (Natalie

Grace and Kelly Roelf, unpublished data). Although we achieved viral titers suitable for efficient transduction, we did not see cJun overexpression in mESCs selected for expression (data not shown).

Previously published literature suggests that perhaps the promoter was the issue.

The pLVX pure plasmid contained a CMV promoter, which was recently shown to be less effective in murine embryonic stem cells. The EF promoter was been shown to be

56 highly active in stem cells (Chung et al. 2002; Hong et al. 2007). Therefore, we designed

a new cloning strategy using the pLVX-EF1α-AcGFP1-C1 Vector, which used the EF-1

α promoter. The successful cloning of the cJun fragments was verified by PCR analysis

and DNA sequencing and their ability to express cJun was confirmed in 293T cells by

Western Blot (All cloning methods and data are reviewed in Appendix I). The new

lentiviral pLVX-EF1α-AcGFP1 expression vectors were successfully transfected into

293T cells, high levels virus assayed confirmed and used to transduce mESCs. The

transduced cells were selected with puromycin at 1.5 μg/ml for 6 days. Although the

transduced cells demonstrated preferential resistance to this antibiotic, the GFP signal

was lost from cells transduced with virus containing the GFP cJun fusion construct. Cells

transduced with GFP alone showed enriched GFP expression. The cell lines were

analyzed by Western blot, PCR and qPCR analysis. When cJun expression was analyzed

by Western blot, only one band appeared at 40 kDa indicating the presence of

endogenous cJun. There was no signal at the predicted size of the GFP-cJun fusion construct (approximately 65 kDa). The qPCR analysis also did not show an increase in the expression of cJun in the transduced sample. (Appendix 1, Figures 8-13).

Our inability to generate stable mESC cells overexpressing cJun led us to revise our experimental design using transient transfection of the same lentiviral constructs. To see if we were able to achieve expression, the pLVX-EF1α-AcGFP1 plasmids containing either GFP cJun, GFP cJun L40/42A, GFP cJun R54A, or GFP alone were transfected into J1 mESC cells. GFP expression in live cell cultures was monitored by microscopy at

12, 24, 36 and 48 hours post transfection. The highest expression of the clones was

57 observed at 36 h post transfection (Figure 18). Similar to mESCs cells transduced with the pLVX-EF1α-AcGFP1- cJun virus and selected with 1.5 μg/ml puromycin, the addition of puromycin for the enrichment of transfection did not increase the expression of the cJun clones. At day10 post 1.5 μg/ml puromycin selection, low transfection was observed and by day 11 no positive green signal was observed in all cJun genotypes. mESCs transduced with viruses encoding all GFP cJun constructs showed abnormal cell growth and the transfected cells were located at the edges of the stem cells clumps or at a single cell stage. The positive control GFP showed normal cells growth and successfully formed a stable cell line (Figure 19).

Figure 18. Expression of GFP cJun, GFP cJun R54A and GFP cJun L40/42A is highest 36 hours post transfection of J1 mESCs. The highest transfection efficiency was observed at 36 h. N= 3. The experiment was performed in triplicate in J1 and B6/BLU stem cell lines.

Figure 19. Selection of J1 mESCs transiently expressing GFP cJun, GFP cJun R54A or GFP cJun L40/42A did not induce stable expression of the cJun fusion proteins. The addition of puromycin for the enrichment of transfected cells did not show higher GFP expression. Transfection was observed at the edges of the colonies. Puromycin selection at 1.5 μg/ml was performed for 10 days. N= 3. The experiment was performed in triplicate in J1 and B6/BLU stem cell lines.

60 The low transfection efficiency of all GFP cJun constructs resulted on limiting the investigation in this study into performing Oct4 expression analysis 36 h after transient transfection. Murine embryonic stem cells were transiently transfected with pLVX-EF1α-

AcGFP1, pLVX-EF1α-AcGFP1 cJun, pLVX-EF1α-AcGFP1 cJun L40/42A or pLVX-

EF1α-AcGFP1 cJun R54A. Expression of the fusion construct was confirmed 36 hours post transfection by GFP fluorescence and immunocytochemistry with cJun antibody

(Abcam #32137). The GFP cJun fusion proteins were predominantly localized in the cell nucleus, where as cells expressing GFP alone was found throughout the cell cytoplasm

(Figure 20).

To test the effect of cJun on Oct4 expression, the transfected cells were subjected to immunocytochemistry with antibodies to Oct4 (Abcam # ab19857) and Oct4A (Cell signaling # 2840s). Data was collected through imaging 15 transfected cells for each treatment. The transfected cells were identified by GFP expression and then the intensity of Oct4A expression was measured using Zeiss microscope and software (Carl Zeiss Inc.,

Thornwood, NY). Finally data from all samples were compared and statistical analysis was performed using SigmaPlot 12 software (Systat Software). Data were collected from the two rounds of experiments in J1 cell lines.

The results showed no difference in overall Oct4 expression, but a significant increase in the Oct4A expression in cells highly transfected with GFP cJun (p=0.0096),

GFP cJun R54A (p=0.0012), or GFP cJun L40/42A (p= 0.0009) when compared to GFP alone (Figures 21-23). When the amount of Oct4A expression was compared among the cJun variants, there was more Oct4A expression in samples that overexpressed GFP cJun

61 and GFP cJun R54 when compared to GFP cJun L40/42A but it was not statistically significant (Figure 23).

Figure 20. GFP cJun and GFP cJun mutants are expressed in J1 murine embryonic stem cells and predominantly localized to the nucleus. Nuclear localization expression of cJun among transfected samples by wild type cJun and the mutant forms. Nuclear and cytoplasm expression of cJun in the transfected control sample and un-transfected cells was observed. Images were taken at 63X. N=2.

Figure 21. GFP cJun expression in J1 embryonic stem cells did not show a measurable increase in total Oct4 protein. Transfected cells with wild type and mutant forms of cJun did not show difference in Oct4 expression. Images were taken at 63X. N=2.

Figure 22. Oct4A expression is increased in J1 embryonic stem cells expressing GFP cJun or GFP cJun variants but not in GFP alone. Increase in the nuclear expression of Oct4A in highly transfected cells with cJun forms. Images were taken at 63X. N=2 for cJun mutants and N=3 for cJun wild type.

Figure 23. Transfected J1 cells with cJun forms show statistically significant increase in Oct4A expression. Significant increase in the Oct4A expression in highly transfected cells with cJun forms. T-test showed statistically significant increase for Oct4A for GFP cJun, GFP cJun R54A, GFP cJun L40/42A in comparison to the negative control, (P=0.0096), (P=0.0012), (P=0.0009) respectively. Total measured area = 1655μM. n= 15.

69 The Role of CJun Signaling on Murine Embryonic Stem Cell Potency

The second aim of this proposal is to see if cJun expression affects the potency of

murine embryonic stem cells. To address this question, we compared alkaline

phosphatase activity and directed differentiation capacity of murine embryonic stem cells

overexpressing GFP vs. GFP cJun, GFP cJun R54A and GFP cJun L40/42A.

Testing the effects of cJun on alkaline phosphatase activity

High levels of alkaline phosphatase are associated with embryonic stem cell

potency (Hirai et al. 2012). To see if cJun signaling affects murine embryonic stem cell

potency, J1 and B6/BLU mESC lines were transfected with pLVX-EF1α-AcGFP1: GFP, pLVX-EF1α-AcGFP1 cJun, pLVX-EF1α-AcGFP1 cJun L40/42A or pLVX-EF1α-

AcGFP1 cJun R54A and tested for alkaline phosphatase activity. GFP Expression was confirmed by fluorescent microscopy 36 hours post transfection (data not shown). The cells were fixed in 4% paraformaldehyde and alkaline phosphatase activity was performed (EMD Millipore, Temecula, CA). Twenty transfected mESC colonies/plasmid were colocalized with GFP and then assayed for alkaline phosphatase activity, in triplicate. The average values were obtained from triplicate and the percentages were calculated out of a total of twenty colonies/genotype.

The results showed mESC colonies overexpressing GFP cJun, GFP R54A or GFP

L40/42A had reduced alkaline phosphatase activity when compared to colonies expressing GFP alone. The addition of GFP cJun resulted in 43% fewer positive alkaline phosphatase colonies when compared to GFP alone (p=0.008; F= 11.39; d.f.= 8). While

70 GFP cJun R54A had 67% less activity and GFP cJun L40/42A was 40% less (p=0.005;

F= 11.39; d.f.= 8)(Figure 24).

B Transfected Average % SEM Statistical differences Plasmids Positive Alkaline compared to GFP alone phosphatase P<0.050

GFP cJun 43% ± 0.577 P=0.008 GFP cJun 40% ± 0.667 P=0.005 L40/42A GFP cJun 67% ± 0.577 - R54A GFP 94% ± 0.333 -

Figure 24. GFP cJun, GFP cJun, GFP cJun L40/42A and GFP cJun R54A decreased alkaline phosphatase activity in J1 murine embryonic stem cells. MESCs were transfected with cJun forms and then subjected to alkaline phosphatase assay post 36h of transfection. Transfected cells were colocalized with GFP and then tested for alkaline phosphatase. A. Images for transfected mESCs post-alkaline phosphatase assay. Red= positive alkaline phosphatase. Green=GFP. Images were taken at 20x. B. Number of positive and negative alkaline phosphatase post transfection with wild type and mutant forms of cJun. n=20. The experiment was performed one time and data collected from triplicate.

72 Examining the effects of cJun on directed differentiation

The pluripotency of embryonic stem cells is often tested by directing their

differentiation into cell types derived from different embryonic germ layers. To see if

overexpression of cJun results in changes in embryonic stem cell potency, cells

overexpressing GFP, GFP cJun, GFP cJun L40/42A or GFP cJun R54A were directed to

differentiate into either pancreatic islet like clusters, which are comprised of cells from

the endoderm and ectoderm lineages, or beating cardiomyocytes, which are derived from

the mesoderm.

Directed differentiation into Pancreatic Islet Like Clusters and Cardiomyocytes

J1 and B6/BLU murine embryonic stem cells were transiently transfected with

pLVX-EF1α-AcGFP1: GFP, pLVX-EF1α-AcGFP1 cJun, pLVX-EF1α-AcGFP1 cJun

L40/42A or pLVX-EF1α-AcGFP1 cJun GFP R54A. Cells were harvested for EB formation 6 hours post transfection. Successful transfection was confirmed by observing

GFP expression by fluorescence microscopy. There were no noticeable differences in EB formation among samples (Figure 25).

Figure 25. Embryoid body formation is not affected by transient transfection of GFP cJun constructs. MESCs plated on gelatin were transiently transfected with each GFP construct. Cells were harvested 6 hours post transfection and plated into ultra low adherent plates in the presence of differentiation. Experiment was performed in J1 and B6/BLU cell lines. Data presented from B6/BLU cell line. N=2.

74 Four days post embryoid body formation; the embryoid bodies were used in the formation of beating cardiomyocytes or pancreatic islet-like clusters differentiation protocol (Stem Cell Technologies, Vancouver CA). Differentiating the transfected cells into beating cardiomyocytes was performed in J1 and B6/BLU cell lines. Yet, no beating cardiomyocytes were observed in any of Jun genotypes and the control after 28 days of observation.

The pancreatic islet-like insulin secreting differentiation protocol was complete after 18 days (Stem Cell Technologies. 2003). Cells were fixed and assayed for insulin and alpha tubulin expression by immunocytochemistry (Figures 26-27). Though all expressed both markers of pancreatic like islet clusters, there were differences in total clusters, cluster size and the morphology of the neuronal projections. These were seen between different forms of cJun. Cells transfected with GFP cJun had a reduced number of pancreatic islet-like clusters when compared to those expressing GFP alone. However, cells expressing the JNK binding mutant GFP cJun L40/42A showed a significant increase in the total number of clusters formed by cells. Statistical analysis of the counted clusters using ANOVA one-way showed the only statistically significant between GFP cJun L40/42A and GFP cJun (p=0.005; F= 6.135; d.f.= 12) (Figure 26-28; Table 6).

There were also notable differences in the size of the clusters from each genotype.

The diameter of each cluster was measured by measuring the length of the longest ends of the insulin secreting portion of each cluster. The sizes sorted into three categories:

≈≤70μM (small), 70μM<350μM (medium) and ≈ ≥ 350μM (big). The results indicated that samples with the addition of GFP cJun showed reduction in the overall size of

75 clusters; statistical analysis showed GFP cJun sample with significant decrease of the

medium clusters formation in comparison to the control GFP and all the other genotypes

(p=0.006; d.f.= 36). Since the addition of GFP cJun resulted in reduction of the total

number and size of pancreatic-Islet like clusters, there is less overall insulin secreting

cells in this sample in comparison to the control and the other cJun mutants. Clusters

derived from GFP cJun L40/40A expressing cells resulted in an increase in the medium and small size clusters when compared to the control GFP. However, the statistical analysis of the data did not show significant differences. GFP cJun L40/42A sample formed the highest number of small clusters, which showed significant increase in compare to the GFP cJun sample (p=0.039; d.f.= 36) (Figure 26, 27, 29; Table 6 ).

Transient expression of cJun also affected the neuronal projections. The neuronal network of these projections was quantified by counting the number of neuronal projection that linked two neighboring clusters together. The experiment was performed in duplicate in J1 and B6/BLU cell lines for each genotype. Statistical analysis with the use of one-way ANOVA showed significant decrease of neuronal formation in GFP cJun genotype in comparison to the control GFP and GFP cJun L40/42A genotype (p=0.044;

F= 4.22; d.f.= 12). Clusters generated from mESCs transfected with GFP cJun R54A showed some reduction in the network formation in comparison to the control, however, no significant difference was observed (Figure 26, 27, 30; Table 6).

Figure 26. Decrease in total number, size and network formation of Pancreatic Islet-like clusters in mouse embryonic stem cells transfected with wild-type cJun. The addition of GFP cJun showed the lowest clusters formation and abnormal alpha tubulin morphology. Images were taken at 10x. Data presented from B6/BLU cell line.

Figure 27. Differences in alpha tubulin and total number of Pancreatic Islet-like clusters derived from J1 mouse embryonic stem cells transfected with wild type GFP cJun. Images were taken at 10X. Data presented from J1 cell line.

Figure 28. Transient Expression of GFP cJun in murine embryonic stem cells reduces the number of pancreatic islet like clusters. The addition of GFP cJun showed a significant decrease of number of clusters formation in comparison to GFP cJun L40/42A. Experiment was performed in J1 and B6/BLU cell line in duplicate. Data presented from both cell lines.

Figure 29. Transient expression of wild type GFP cJun in murine embryonic stem cells resulted in significant decrease in the size of pancreatic islet like clusters. The addition of GFP cJun showed a significant decrease in the medium size of clusters in comparison to all the other genotypes. Additionally, the GFP cJun L40/42A showed increase in the small clusters formation. Small: ≈≤70μM. Medium: ≈>70μM<350μM. Big: ≈ ≥ 350μM. Experiment was performed in J1 and B6/BLU cell line in duplicate. Data presented from both cell lines.

Figure 30. Transient expression of wild type GFP cJun in murine embryonic stem cells resulted in significant decrease of network formation between clusters in comparison to GFP cJun L40/42A and GFP genotypes. The addition of GFP cJun showed a significant decrease of network formation between clusters in comparison to GFP cJun L40/42A and GFP genotypes. The connection between two clusters was counted as a network. Experiment was performed in J1 and B6/BLU cell line in duplicate. Data presented from both cell lines.

Table 6. Differences in total number, size and network formation in pancreatic Islet-Like clusters post the addition of different forms of cJun. Data was collected from J1 and B6/BLU cell lines Experiment was performed in duplicate.

Averages GFP GFP cJun GFP cJun GFP cJun L40/42A R54A

Average total number of 58 312.75 213.5 212 colonies Average number of small 40.5 134.25 90.5 48.25 colonies Average number of Medium 14.5 150.25 108.75 132.25 colonies Average number of Large 2 27 14.25 33.75 colonies Average number of network 23.5 425.25 229 422.75 between colonies

82 The Role of JNK Signaling on Murine Embryonic Stem Cell Potency

The third aim of this proposal is to see if JNK signaling expression affects the

potency of murine embryonic stem cells. To address this question, we compared alkaline

phosphatase activity and directed differentiation capacity of murine embryonic stem cells

treated with the JNK chemical activator anisomycin and the JNK chemical inhibitor

SP600125. As both are reconstituted in DMSO, a DMSO only control was used.

Testing the effects of modulating JNK signaling on alkaline phosphatase activity

Murine embryonic stem cell lines J1 and B6/BLU were cultured in the presence

of 10 ng/ml anisomycin, 10 µM SP600125, or DMSO alone for two days. Cells were

fixed in 4% paraformaldehyde forty eight hours post chemical treatments and an alkaline

phosphatase assay was performed (EMD Millipore, Temecula, CA). The total number of

positive and negative alkaline phosphatase colonies was counted and the intensity of

color noted. One-way ANOVA showed treatment with 10 μM SP600125 resulted in

significant increase (p=0.020; F= 18.7; d.f.=3) of positive alkaline phosphatase colonies

(darker red= more pluripotency state) in comparison to cells treated with anisomycin or

DMSO, suggesting that the down-regulation of JNK pathway leads to an increase in

mESC pluripotency (Figure 31). Anisomycin treatment at 10 ng/ml showed a smaller but

still significant increase in alkaline phosphatase expressing colonies, however only the

cells treated with 10 µM SP600125 were consistently a dark red (Figure 31).

Murine embryonic stem cells are cultured in the presence of the leukemia inhibitory factor (LIF) cytokine to maintain pluripotency. The significant increase in

83 alkaline phosphatase activity seen in cells treated with the 10 μM SP600125 prompted us to see if the SP600125 was sufficient to maintain potency. Cells were cultured for 6 days in either LIF alone, LIF and 10 μM SP600125, or 10 μM SP600125; fixed with 4% paraformaldehyde; and an alkaline phosphatase assay was performed (EMD Millipore,

Temecula, CA). Images were taken by light microscope for J1 and B6/BLU cell lines that had the same earlier treatments for four days. Data was analyzed by one way ANOVA.

Cells grown in 10 μM SP600125 inhibitor without LIF showed the highest positive alkaline phosphatase colonies with the darkest red color indicating less differentiation initiation (p=0.011; F= 86.7; d.f.= 2) (Figure 32). The data showed the maintenance of mESCs morphology in all samples (Figure 33).

Figure 31. SP600125 treatment of murine embryonic stem cells resulted in the highest formation of undifferentiated mouse embryonic stem cells colonies. A. Mouse embryonic stem cells were cultured in the presence of SP600125 on gelatin plates for two days. Cells were fixed and alkaline phosphatase (AP) assay was performed. Images were taken under light microscope and positive AP was counted. Values obtained from 2 samples/treatment including 440 partly differentiated colonies. B. Represent light microscope images were taken at 4X. Red= positive alkaline phosphatase. Experiment was performed once in triplicate.

Figure 32. Treatment of embryonic stem cells with SP600125 ± LIF resulted in the highest undifferentiated mouse embryonic stem cells colonies 6 days post treatment. A. Mouse embryonic stem cells were cultured in the presence of SP600125 for 6 days. Cells were fixed and alkaline phosphatase (AP) assay was performed. Images were taken under light microscope and positive AP was counted. Values obtained from 2 samples/treatment including 440 partly differentiated colonies. B. Represent light microscope images were taken at 4X. Red= positive alkaline phosphatase. Experiment was performed twice in triplicate.

Figure 33. Mouse embryonic stem cells morphology is maintained after 4 days of 10 μM SP600125 ± LIF treatments. Images were taken by light microscope at 20X. N=2. Experiment was performed twice in triplicate. Experiment was performed in J1 and B6/BLU cell lines.

87 Testing the effects of modulating JNK on the differentiation of mESCs

The dramatic effect of SP600125 on alkaline phosphatase activity suggested JNK

activity could also affect directed differentiation. J1 murine embryonic stem cells were

treated with 10 ng/ml anisomycin, 10 µM SP600125, or DMSO for four days; harvested;

and plated into ultra low adherent plates in the presence of differentiation media to

initiate embryoid body formation. After four days, differences in morphology were noted

among the different treatments. Samples with the addition of SP600125 showed

increasing in clumps of embryoid bodies formation and the lowest number of circular

morphology formation compare to the other two treatments. MESCs treated with

anisomycin had higher circular embryoid bodies formation than clumps formation

(Figure 34,35). We next tested the ability of these embryoid bodies to form either

pancreatic islet –like insulin secreting clusters or beating cardiomyocytes.

Directed differentiation into Pancreatic islet like clusters

Four days post embryoid body formation; the embryoid bodies were used in a

pancreatic islet-like differentiation protocol (Stem Cell Technologies, Vancouver CA).

Eighteen days later, pancreatic Islet-Like cluster morphology was observed in the culture dishes. Cells were fixed and assayed for insulin and alpha tubulin by immunocytochemistry. The results showed a significant reduction in insulin secretion in

embryoid body treated with anisomycin in comparison to SP600125 or DMSO. All

treatments were able to generate neurons, but differences in morphology were observed

among samples (Figure 36).

Figure 34. Treatment of J1 murine embryonic stem cells with anisomycin or SP600125 affects the embryoid body morphology in J1cell line. Embryoid body treated with SP600125 at 10 µM showed high clumps morphology and low circular formation. N=2. Experiment was performed twice in triplicate.

Figure 35. Treatment of J1 murine embryonic stem cells with anisomycin or SP600125 affects the amount of circular and clumped embryoid bodies. A- embryoid body treated with SP600125 showed the lowest circular formation. B- embryoid body treated with SP600125 showed the highest clumps formation. Experiment was performed twice in triplicate.

Figure 36. Pancreatic Islet-like clusters derived from J1 murine embryonic stem cells treated with 10 ng/ml anisomycin at or 10 µM SP600125 show different levels of insulin expression and neuronal morphology. The anisomycin treatment showed down-regulation of Insulin secretion in comparison to SP600125 and the control treatment and different neurons morphology among the different treatments. Images were taken at 20x.

90 The differentiation experiment was repeated in the J1 and B6/BLU cell lines. Data for the second round of the experiment was analyzed through measuring the number of clusters, size and neuronal formation (for details see page 74,75). The results showed treatment with 10 ng/ml of anisomycin in J1 and B6/BLU cell lines resulted in a decrease of the total cluster number and size when compared to the control samples. Treatment with 10 µM SP600125 resulted in some increase of the total numbers of clusters and it showed increase in the formation of small size clusters (Figure 37-40; Table 67).

However, statistical analysis of the data by performing one-way ANOVA did not show significant differences (p=0.3813; F= 1.333; d.f.= 4).

In this round of experiment, more significant differences were observed in neuronal formation. In both cell lines, we observed an obvious reduction in neuronal network formation between clusters. The neuronal network of these projections was quantified by counting the number of neuronal projections that linked two neighboring clusters together. Samples treated with 10 ng/ml anisomycin showed dramatically impaired neuronal formation, while cells treated with 10 µM SP600125 had a noticeable increase (Figure 37,38 and 41; Table 7). However, statistical analyze by performing one- way ANOVA did not showe significant differences among samples (p=0.3652; F= 1.4; d.f.= 4). In the B6/BLU cell line neuronal formation was dramatically impaired.

J1 cell line did not show the same significant decrease in the insulin secreting in the10 ng/ml anisomycin sample in comparison to the results from the first round (Figure

42). However, the decrease in the total clusters number and size indicated decreasing the total insulin secretion in the 10 ng/ml anisomycin sample in comparison to the control.

Figure 37. Chemical modulation of JNK activity in J1 mESCs affects pancreatic islet like cluster formation. Cells treated with 10 ng/ml anisomycin showed smaller cluster and neuronal network formation when compared to cells treated with 10 µM SP600125 or DMSO treatment. It also showed different alpha-tubulin morphology. Images were taken at 2.4x.

. Figure 38. Chemical modulation of JNK activity in B6/BLU mESCs affects pancreatic islet like cluster formation. Cells treated with 10 ng/ml anisomycin showed smaller cluster and compromised neuron formation when compared to cells treated with 10 µ M SP600125 or DMSO treatment. It also showed different alpha-tubulin morphology. Images were taken at 20x. Experiment was performed once in this cell line. 93

Figure 39. Chemical modulation of JNK activity in murine embryonic stem cells affects the total number of pancreatic Islet- Like clusters. Data was collected from J1 and B6/BLU cell lines. Experiment was performed in two cell lines.

Figure 40. Chemical modulation of JNK activity in murine embryonic stem cells affects the size of pancreatic Islet-Like clusters. The addition of anisomycin and SP600125 resulted in changing the size of the clusters. Small: ≈≤70 μM. Medium: ≈>70 μM<350 μM. Big: ≈ ≥ 350μM. Experiment was performed in J1 and B6/BLU cell line in duplicate. Data presented from both cell lines.

Figure 41. Chemical modulation of JNK activity in murine embryonic stem cells affects neuronal network formation between pancreatic Islet-Like clusters. The addition of anisomycin and SP600125 resulted in changes in the network formation between clusters among the different samples. The connection between two clusters was counted as a network. Experiment was performed in J1 and B6/BLU cell line in duplicate. Data presented from both cell lines.

Table 7. Differences in total number, size and network formation in pancreatic Islet-Like clusters derived from mESCs treated with SP600125 or anisomycin. Data was collected from J1 and B6/BLU cell lines Experiment was performed in duplicate.

Averages 10 µM 10 ng/ml DMSO No SP600125 anisomycin treatment

Average total number of 381 206 266 316 colonies Average number of small 365 171 251 295 colonies Average number of Medium 14 29 12 17 colonies Average number of Large 2.6 5.3 2 4 colonies Average number of network 448 201 241 323 between colonies

Figure 42. . J1 mESCs can produce insulin secreting pancreatic islet like clusters after chemical modulation of JNK activity. Pancreatic Islet-Like clusters that were formed after anisomycin and SP600125 treatments showed insulin secretion. Images were taken at 20x.

98 Directed differentiation into Cardiomyocytes

Embryoid bodies formed from cells treated with either 10 ng/ml anisomycin or 10

µM SP600125 were plated to tissue culture dishes coated with 0.1% gelatin and cultured

in differentiation media. The number of beating cardiomyocytes/sample was counted

each day for ten days. The results showed differences in the formation of beating clumps

between the treatments. Samples treated with 10 µM SP600125 had the most beating clumps at the earliest time point, while no beating clumps were observed in the sample treated with 10 ng/ml anisomycin until the final day of the experiment (Table 8).

Table 8. Treatment with SP600125 showed the highest number of beating cardiomyocytes. Embryoid body treated with SP600125 had the highest colonies formation and samples treated with anisomycin had the lowest. J1 cell line. N=1.

Treatment Number of Number of beating beating colonies colonies in 10 days in 8 days (192h) (240h)

DMSO (control) 1 3 SP600125 at 10 µm 6 9 Anisomycin at 10 ng/ml 0 1

99 To test the role of JNK activity on the mouse embryonic stem cell potency, the addition of the chemical treatments was performed followed by the formation of embryoid bodies and differentiation into cardiomyocyte colonies. MESCs were treated with 10 µM SP600125, 10 µM SP600125 + LIF, or DMSO + LIF. All samples were able to form embryoid bodies (Figure 43). The same samples were differentiated into cardiomyocyte beating colonies. The results showed all samples that had the addition of

SP600125 ± LIF and the control were able to form beating colonies (Figure 44). The same results were observed in J1 and B6/BLU cell lines.

Figure 43. Murine embryonic stem cells treated with SP600125 ± LIF for 2 days successfully formed Embryoid body. Images were taken at 2.4X post 24h of Embryoid body initiation. Experiment was performed twice in triplicate. J1 cell line.

100

Figure 44. Murine embryonic stem cells treated with 10 µM SP600125 ± LIF successfully formed cardiomyocyte beating colonies. Treatment with SP600125 at 10 µM ± LIF formed beating colonies. Experiment was performed twice in triplicate. J1 cell line.

101

DISCUSSION

The goal of this study was to determine the role of JNK/cJun signaling on Oct4 expression and potency of murine embryonic stem cells. Our results provide strong support for the hypothesis that both cJun and JNK directly regulate Oct4 expression and stem cell potency, however it is likely they do so through multiple regulatory mechanisms.

CJun and JNK Signaling Affects Oct4 Expression

The effect of modulating the JNK/cJun pathway on Oct4 expression in murine embryonic stem cells was examined in two different mESC cell lines by chemical treatments with anisomycin and SP600125 and through transient transfection of GFP cJun expression constructs. Though antibodies directed to all Oct4 isoforms did not show differences in Oct4 expression, an increase in the Oct4A isoform was seen in cells transfected with each GFP cJun construct or treated with anisomycin, which we showed increased the levels of endogenous phosphorylated cJun. The addition of the chemical

SP600125 at 50 μM also showed increased expression of Oct4A. These results indicate that while cJun activation through JNK phosphorylation can regulate expression, there may be additional, independent mechanisms regulating this phenomenon. Some potential models have recently been shown in the literature. A study by Bryn et al

(2013) showed down-regulation of Oct4 expression through the SAPK/JNK regulation of heat shock factor (HSF1) in human embryonic stem cells. Additionally, they were

102 able to increase phosphorylation of HSF1, and therefore Oct4 expression, through inhibition of JNK using SP600125 (Byun et al. 2013). Another study describes a cJun regulation mechanism that is not dependent on MAPK activity. Blau et al (2012) demonstrated that cJun could be translationally regulated by mTOR cytoskeleton- dependent pathway that leads to accumulation of cJun in glioma cell lines. That we see an increase in Oct4 expression in the presence of anisomycin fits the model, as anisomycin is a suppressor of rapamycin sensitive translation, thus functioning to increase mTOR and eIF4A translation from IRES sequences (von Manteuffel et al.1996).

CJun/JNK Activity Effects MESC Potency

As Oct4 is best known as a regulator of mESC potency, it seemed likely that if cJun/JNK regulated Oct4, they would also affect the potency of murine embryonic stem cells. Alkaline phosphatase expression is a classic marker of murine embryonic stem cell potency. Murine embryonic stem cells transiently expressing GFP cJun, GFP cJun

L40/42A or GFP cJun R54A all showed less alkaline phosphatase activity when compared to GFP alone, with GFP cJun L40/42A showing the most significant reduction in alkaline phosphatase activity (40 % Positive alkaline phosphatase) followed by GFP cJun (43%). Transfection with GFP cJun R54A resulted in the most activity (67%). To our knowledge, this study is the first evidence that cJun to murine embryonic stem cell potency.

103 Support for a role for JNK signaling in stem cell potency was evident when mESCs were treated with the SP600125. Standard murine embryonic stem cell culture conditions require the presence of murine leukemia inhibitory factor (LIF) to maintain the JAK/STAT signaling required for pluripotency. The addition of 10 μM SP600125 with or without LIF resulted in smaller colony formation and lower cell number, but there was a significant increase in alkaline phosphatase expression. Similar results were recently reported by Yao et al 2014, who also demonstrated withdrawing LIF with the addition of SP600125 suppressed the differentiation process, but not through cJun.

Their work showed JNK activity can inhibit the transcription and transactivation of the transcription factor KIF4, a positive regulator of pluripotency gene expression. They also showed a negative role for SP600125 the reprograming to pluripotent stem cells.

Testing proliferation rates of mESCs treated under these conditions could inform whether cJun’s role in cell cycle progression could account for the smaller colony size.

The loss of pluripotency predicted by the alkaline phosphatase assay was seen in the directed differentiation experiments performed under similar conditions. Murine embryonic stem cells transfected with wild type GFP cJun exhibited a decrease in total number and size of pancreatic islet-like insulin secreting clusters in comparison to the negative control and the other cJun mutants. Cells transfected with wild type cJun also showed deficient and short alpha tubulin morphology formation and significant reduction in neuronal network formation. The reduction in the number and size also indicated the reduction in the total insulin formation in this sample. Transient expression of the GFP cJun L40/42A mutant showed significant increase in the total

104 number of clusters, the formation of small pancreatic islet-like clusters and network formation in comparison to sample that had the addition of wild type cJun, suggesting transcriptional activation through JNK is significant. When either J1 or B6/BLU mESCs were treated with 10 ng/ml anisomycin or 10 μM SP600125 before directed differentiation to pancreatic islet-like clusters, cells treated with anisomycin had less total islet-like clusters. Those that did develop were of a smaller size, had impaired neuronal development and less overall insulin production. The addition of SP600125 showed an obvious increase the total number, small size and the network formation of the clusters in comparison to the controls and the anisomycin sample. That we see parallel differentiation results in cells overexpressing forms of cJun with differences in their ability to be phosphorylated by JNK suggest cJun is at least one downstream target of SP600125 this process.

These results support other work examining the roles of JNK in development.

Several studies showed that the inhibition of JNK promotes the differentiation process.

One study showed rapid proliferation and abnormal differentiation in mesodermal lineage in vitro and ectoderm development in vivo in JNK-/- mESCs (Xu and Davis

2010), and a second study showed that the use of SP600125 and ERK inhibitor resulted in increasing osteogenic differentiation of bone marrow stromal cells, suggesting a possible treatment for bone regeneration (Doan et al. 2012). Another study showed initiating of differentiation of epidermal keratinocytes as as a result of SP600125, giving a possible differentiation treatment for psoriasis (Gazel et al. 2006).

105 That we see parallel differentiation results in cells overexpressing forms of cJun with differences in their ability to be phosphorylated by JNK suggest cJun is at least one downstream target of SP600125 this process. Overexpression of GFP cJun or GFP cJun

R54A reduces the differentiation to pancreatic islet-like clusters while GFP cJun

L40/42A increases the differentiation process. It has previously been shown that the activation of cJun through JNK by the addition of human amylin induces apoptosis in pancreatic islet-β cells, which might explain the decrease in the total number of clusters in samples with increased cJun expression that was observed in this study (Zhang et al.

2002). JNK activity acts as a negative feedback regulator of the insulin signal in mouse embryo fibroblasts, 3T3-L1 adipocytes, and 32DIR cells by phosphorylation of rat/mouse Irs1 at Ser307 (Lee et al. 2003). Lee et al. (2003) proposed the activation of

JNK would result in preventing the activation of insulin, thus inhibiting JNK reduce insulin resistance, suggesting that could be a possible therapeutic treatment for diabetes.

Another study showed that JNK activation in pancreatic β-cells in transgenic mice resulted in insulin resistance in these cells, but did not promote β-cell death by inducing glucose intolerance (Lanuza-Masdeu et al. 2013). Additionally, JNK has been shown to play roles in neuronal function (Reviewed in Coffey 2004) and Oliva et al (2006) showed the inhibition of JNK by the addition of SP600125 resulted in inhibiting axonogenesis in embryonic day 18 rat hippocampal neurons. Yet, it did not prevent their differentiation into dendrites (Oliva et al. 2006).

We also showed evidence that JNK activity can affect beating cardiomyocyte formation. Murine embryonic stem cells treated with SP600125 showed 6 beating

106 clumps compares to one clump in the control, whereas no cardiomyocyte differentiation was observed in sample treated with anisomycin at day 8 post the starting of the differentiation process. We were not able to generate beating clumps in sample transfected with GFP or GFP cJun. We suspect this is due to a reagent change: the fetal bovine serum we historically used for this experiment is no longer available by the producer company.

Differences in the Subcellular Localization of CJun Between 293T and MESCs

Although the main focus in this study was to examine the role of cJun on the regulation of Oct4, differences in the subcellular localization of cJun were discovered between 293T cells and mESCs. The data showed cJun present predominantly in the nucleus of dividing 293T cells, while cJun was present in both the nucleus and cytoplasm of mESCs (Figure 15). Western blot analysis showed higher cytoplasmic localization of cJun after anisomycin treatment at 50 ng/ml in mESCs and 293T (Figure

7-14). The differences in the localization of cJun between the 293T and mESCs indicate different regulation and function of cJun in different cell types.

Subcellular localization differences were also observed for phosphorylated cJun at S63 and S73 for 293T and mESCs. In 293T cells, immunocytochemistry data after the activation of JNK/cJun pathway for thirty minutes showed p-cJun S63 to be localized mostly in the nucleus, and the inhibition of JNK showed more cytoplasmic localization (Figure 4). In mESCs, p-cJun S63 was expressed exclusively in the cytoplasm except after treatment with anisomycin at 50 ng/ml for two hours, which

107 showed cytoplasmic and nuclear localization (Figure 12). Western blot showed higher protein levels of p-cJun S63 in the cytoplasm and the nucleus after treatment with anisomycin at 50 ng/ml for two hours in mESCs (Figure 14). 293T cells also showed an increase in p-cJun S63, but only in the cytoplasm (Figure 7). Cytoplasmic localization of p-cJun S63 after treatment with anisomycin at 50 ng/ml suggests that the highly activated form of p-cJun S63 might be prevented from entering the nucleus of the cells. Additionally, while 293T were treated with chemicals for thirty minutes before performing immunocytochemistry; it was treated for two hours before performing

Western blot; which explain the differences in the subcellular localization of cJun between Western blot and immunocytochemistry.

Immunocytochemistry data also showed the subcellular localization of the p- cJun S73 with different pattern than p-cJun S63 in mESCs. The expression of p-cJun

S73 was mostly nuclear localized after activating and inhibiting the JNK/cJun pathway

(Figure 13). The experiment needs to be repeated at an identical time point, but the results suggested there might be a different phosphorylation pattern of cJun that could have specific biological effects. Zhang et al. 2002 showed the phosphorylation of cJun at S63 but not S73 induces apoptosis in the β – cells. Others have shown the accumulation of cJun in retinal cells induced apoptosis (Chiarini et al. 2002). It is well established that the complexity of cJun in the regulation of cell growth and apoptosis, particularly the activation of JNK/cJun, results in differing cellular responses, and that this results is largely dependent on signaling pathways, chromatin domains and activation of other proteins (reviewed in Lebba and Bohmann 1999; Shaulin and Karin

108 2002). However, there is evidence that links the presence of cJun in the cytoplasm of retinal cells to apoptosis (Chiarini et al. 2002). A more recent study contradicted the earlier study by showing that the phosphorylated forms of cJun at S63 and S73 in neurons was negative for TUNEL staining, indicating the absence of the apoptosis with cJun activation (Thakur et al. 2007). The role of cJun in apoptosis might explain our inability to generate mESCs stably expressing the GFP cJun constructs. We attempted to generate mouse embryonic stem cell lines expressing GFP, GFP cJun, GFP cJun

L40/42A and GFP cJun R54A using a lentiviral system twice. We were only able to generate GFP expressing cells using this system. Cells transduced with any of the GFP cJun viruses lost GFP expression even in the presence of puromycin (see Appendix 1 for details). Performing transient transfection with antibiotic selection showed the same abnormal cell growth. These experiments, as well as our results demonstrating anisomycin can reduce cell viability, suggests that modulating the expression of cJun in mESCs had disturbed the biology of the cells.

Conclusions

Oct4 is essential for the self-renewal of the stem cells and it is implicated in many cancers. Altogether, the data in this study supports the hypothesis that cJun/JNK signaling affects mESC potency. However, we did not predict murine stem cell potency would be reduced in the presence of cJun/JNK activity considering the Oct4A transcript is upregulated under these conditions. Oct4A is thought to be solely responsible for pluripotency until two additional human isoforms were characterized: Oct4B-and

109 Oct4B1 (Atlasi et al. 2008). Atlasi et al. 2008 and others have shown data suggesting

Oct4B1 as a stemness marker in human embryonic stem cells (hESCs). Papamichos et al. 2009 identified Oct4B1 in 59 hESCs lines (Papamichos et al. 2009). Oct4B and

Oct4B1 isoforms have not been described in mESCs. Further investigation for Oct4 isoforms in mESCs is an important step toward understanding the role of each isoform in the mESCs potency and differentiation.

Additionally, performing apoptosis and cell cycle assays post modulating

JNK/cJun will be an important step toward understanding the direct affect of the JNK pathway on the mESCs fate. Testing the ability of mESCs to form the three germ layers after modulating JNK/cJun will be another crucial step toward understanding its effect on the potency of murine embryonic stem cells.

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120 APPENDIX A. GENERATION of pLVX-EF1α-AcGFP1-CJUN CONSTRUCTS.

Materials and Methods

NCBI Primer Blast (Ye et al. 2012) was used to design PCR primers developed to amplify the murine cJun coding sequence (NCBI accession number NC_000070.6) previously cloned into RCASA expression vectors (Sprowles and Wisdom 2003). The

BamH1 restriction enzyme cut site was added to both the forward primer and the reverse primer to generate the final PCR primer pair; Forward : 5'-GCG GGC CCG GGA TCC

ATG ACT GCA AAG ATG GAA ACG ACC TTC TAC-3’ ;Reverse : 5'-AGA TCC

GGT GGA TCC TCA AAA CGT TTG CAA CTG CTG CGT-3’.

The PCR reaction components were as follows: 1ng template (RCASA cJun, cJun

L40/42A or cJun R54), 12.5 µl 2x high fidelity master mix (Clontech, Mountain View,

CA), 0.25 µM forward primer, 0.25 µM reverse primer and nuclease free water for a final volume of 25 µl. A reaction without template DNA was used as a negative control. The reaction was run on an MJ Research PCT-100 thermal cycler (Thermo Scientific,

Pittsburgh, PA). Reaction conditions were as follows: initial denaturation: 94ºC, 2 minutes; 30 x (melting: 94ºC, 30 sec.; annealing: 75.5ºC, 30 sec.; extension: 73ºC, 5 sec.); final extension: 65ºC, 7 minutes.

5 µl of each PCR product was analyzed by agarose gel electrophoresis on a 0.7 % agarose gel run at 122 V for 60 minutes. A GeneRuler 1 kb DNA Ladder (Thermo scientific, Rockford, IL) was loaded as a size reference. The gel was stained in ethidium

121 bromide for 10 minutes, destained in water for 10 minutes, then imaged with

Alphalmager HP Gel imaging system (Alpha Innotech, San Leandro, CA).

The remainder of the PCR products (20 µl each) were purified using a QIAquick

PCR purification kit (Qlagen, Valencia, CA). For each genotype, 100 µl of buffer PB

was added to 20 µl of the PCR product. A QIAquick purification column was placed in a

collection tube (provided) and the mixture was transferred to the column. The column

was centrifuged at 16,000 x g for one minute and the flow through was discarded. The

column was then washed by adding 750 µl of buffer PE and centrifuging at 16,000 x g

for one minute. The flow through was discarded and the column was placed back in the

collection tube and centrifuged at 16,000 x g for one minute to remove remaining liquid.

The DNA was then eluted into a sterile 1.5 ml microfuge tube by adding 50 µl of elution

buffer to the column, incubating at room temperature for five minutes, and centrifuging at

16,000 x g for one minute. The concentration of eluted DNA was measured using a ND-

10000 Nanodrop spectrophotometer (Fisher Scientific, San Diego, CA). The purified

PCR product was verified for purity and size by agarose gel electrophoresis by running

100 ng of each genotype on a 0.7 % agarose gel at 122 V for 60 minutes. The gel was stained and imaged as described above.

Each of the purified cJun fragments was then cloned into the pGEM®-T Easy

Vector (Promega, Madison, WI). The ligation of the cJun fragment and pGEM-T was performed at a 1:1 molar ratio of insert: vector in 10 µl reaction including: 50 ng of vector, 50 ng insert, 1 µl of T4 DNA ligase, 5 µl of 2X rapid ligation buffer and deionized water for a final volume of 10 µl. A similar reaction without insert vector was

122 included as a background control. The ligation reactions were incubated overnight. 2 µl

of the ligation reaction was added to 50 µl of JM109 E. coli cells (Promega , Madison,

WI) into a sterile 1.5 ml microfuge tube and incubated for 20 minutes on ice. The cells

were heat-shocked for 50 seconds at 42ºC, then incubated on ice for 1-2 minutes. 950 µl

of SOC medium was added to the cells, and the mixture was incubated for one hour at

37ºC on a shaking tray at 3 x g. After the incubation, 100 µl of the transformed cells

were plated on Luria Broth (LB) agar (SIGMA-ALDRICH®, Louis, MO) plates

containing 50 µg/ml ampicillin, 0.1 mM IPTG and 40 µg/ml X-GAL. The reaction was incubated at 37ºC overnight.

The pGEM® cJun clones was tested with single colony PCR and restriction enzyme digest for each of the cJun genotypes. For each of the cJun genotypes, five randomly picked colonies were tested with single-colony PCR using the cJun primers listed above. The PCR reaction components were as follows: 12.5 µl 2x master mix

(Promega, Madison, WI), 0.25 μM forward primer, 0.25 μM reverse primer, 12 µl of

nuclease free water, and a swab of a white colony. A reaction without a colony swab was

included as a negative control. The thermal cycler was programmed as follows: initial

denaturation: 95ºC, 5 min; 30 x (melting: 95ºC, 30 sec.; annealing: 75.5ºC, 1 min.;

extension: 73ºC, 5 sec.); final extension: 65ºC, 7 minutes. The PCR products were

analyzed with gel electrophoresis as described above. All tested colonies were further

grown in 6 ml LB media in the presence of 50 μg/ml ampicillin overnight at 37ºC. For

restriction enzyme digestion, the DNA from each bacterial culture that tested positive in

the single-colony PCR was purified with the Wizard® Plus SV Miniprep DNA

123 Purification System (Promega, Madison, WI). Cells from each colony were expanded in

six ml LB media overnight at 37ºC in the presence of 50 μg/ml ampicillin. The bacterial

cultures were centrifuged for 5 minutes at 10,000 x g in a tabletop centrifuge and the

supernatant was discarded. The cell pellet was resuspended in 250 μl of cell suspension

solution and transferred to a sterile 1.5 ml microfuge tube. Cell was lysed with 250 μl cell

lysis solution for 5 minutes. 10 μl of alkaline protease was then added and the mixture

was incubated for 5 minutes, followed by the addition of 350 μl neutralization solution.

The mixture was immediately inverted four times and centrifuged at 13,000 x g for 10

minutes. The cleared lysate was transferred to a spin column (provided) and centrifuged at 13,000 x g for one minute. The flow through was discarded and the column was washed by adding 750 μl wash solution, followed by centrifuging for one minute. The flow through was discarded, and the wash step was repeated with 250 µl of the washing

solution. The spin column was then transferred to a sterile 1.5 ml microfuge tube and the

plasmid DNA was eluted in 100 μl of nuclease free water. The DNA concentration was

measured on the ND-10000 Nanodrop spectrophotometer (Fisher Scientific, San Diego,

CA).

Each plasmid was then screened by restriction enzyme digestion in a 20 µl

reaction which contained 500 ng of purified DNA, 30 units BamH1 restriction enzyme

(Promega, Madison, WI), 2 µl buffer E (Promega, Madison, WI) and nucleus free water

to make up the final volume of 20 µl. Digestion reactions were incubated at 37ºC for 3

hours. The digested products were analyzed with gel electrophoresis as described earlier.

To further confirm the presence of the correct insert, plasmids that tested positive with

124 the single colony PCR and restriction enzyme digestion were sent for sequencing by

Sequetech DNA Sequencing Service and the sequences were confirmed using NCBI

BLAST (Camacho et al. 2009).

Confirmed cJun pGEM-T Easy sequences were cloned into the pLVX-EF1α-

AcGFP1-C1 lentiviral expression vector (Clontech, Mountain View, CA). The

Restriction pLVX-EF1α-AcGFP1-C1 vector was linearized with BamH1 in a reaction

containing 1000 ng of pLVX-EF1α-AcGFP1-C1 vector, 100 units BamH1, 2 µl buffer E

and nuclease free water to make up the final volume of 20 µl. The reaction was incubated

at 37 ºC for 3 hours. After the incubation time, the mixture treated with 0.06 units of Calf

intestinal alkaline phosphatase (CIAP) (Promega, Madison, WI) for minutes at 37

ºC. 100 µl of the linearized and CIAP treated pLVX-EF1α-AcGFP1-C1 vector was

analyzed with gel electrophoresis and purified using a QIAquick PCR purification kit

(see above for detailed methods).

The cJun fragments were isolated from the pGEM-T cJun clones by BAMH1 digestion. The digestion contained of 2000 ng of plasmid DNA, 30 units BamH1 restriction enzyme, 2.5 µl of buffer E and nuclease free water to make up the final volume of 25 µl. The reaction was incubated at 37ºC for 3 hours. The digested products were purified with QIAquick PCR purification kit and analyzed with gel electrophoresis as described above.

The cJun fragments were ligated into the pLVX-EF1α-AcGFP1- at a 3:1 insert: vector molar ratio in a reaction containing of 100 ng of vector:32.5 ng insert, 1 μl of T4

DNA ligase (Promega, Madison, WI), 10 µl of 2X rapid ligation buffer (Promega,

125 Madison, WI) and deionized water for a final volume of 20 µl. A reaction without DNA

was used as a negative control. The ligation reactions were incubated overnight at 4ºC

and the resulting plasmids were transformed into JM109 E. coli cells as described

previously.

To identify clones with the cJun insert, 19 randomly picked white colonies for

each genotype were expanded in 6 ml LB media in the presence of 50 µg/ml ampicillin.

The cultures were grown overnight in a shaker at 37ºC and plasmid DNA from these cultures was purified using Wizard® Plus SV Minipreps DNA Purification Systems (see above for detailed methods). BamH1 restriction enzyme digestion was performed as described above. A secondary restriction enzyme digest was performed to identify cJun inserts in the correct orientation. The reaction consisted of 300 ng plasmid DNA, 30 units

Kpn1 (Promega, Madison, WI), 30 units Pst1 (Promega, Madison, WI), 2 µl buffer C

(Promega, Madison, WI) and nuclease free water for a final volume of 20 µl. The reactions were incubated at 37ºC for 3 hours and the digested products were analyzed with gel electrophoresis. Positive cJun clones for each genotype were stored by mixing

500 µl of the transformed E. coli with 500 µl glycerol (SIGMA-ALDRICH®, Louis,

MO) and frozen in -80ºC.

Positive cJun pLVX-EF1α-AcGFP1-C1 clones were sequenced by Sequetech

DNA Sequencing Service (Mountain View, CA) and the sequences were confirmed using

NCBI BLAST.

The verified cJun clones were amplified with NucleoBond® Xtra Midi plus EF

(Clontech, Mountain View, CA) following the manufacturer’s instructions. Positive

126 transformed E. coli cells were thawed from -80 ºC and a swab from each vial of the

different genotypes was expanded in 3 ml of LB media in the presence of 50 µg/ml

ampicillin for 6 hours in a shaker at 37ºC. The culture was further expanded by

transferring 1.5 ml of the culture into 100 ml of LB media in the presence of 50 µg/ml

ampicillin and incubated for 16 hours in a shaker at 37ºC. The cultures were then spun

down at 6000 x g for 15 minutes. Each cell pellet was re-suspended in 8 ml of buffer

RES-EF and transferred into 50 ml conical tube. The sample was lysed with 8 ml of buffer LYS-EF for 5 minutes then neutralized by the addition of 8 ml of buffer NEU-EF

on ice for 5 minutes. The lysate was centrifuged at 5000 x g for 10 minutes and the clear

lysate was loaded into the equilibrated NucleoBond® Xtra column filter (provided), and

allowed to flow through the column with gravity. The column was washed with 5 ml of

buffer FIL-EF and the NucleoBond® Xtra column filter was discarded. A second wash

with 35 ml of buffer ENDO-EF and a third wash with 15 ml of buffer WASH-EF was

performed. For all wash steps the column was emptied by gravity and the flow through

was discarded. The plasmid DNA was eluted in 5 ml of buffer ELU-EF and the DNA was

collected in 15 ml conical tube. The DNA was precipitated with 3.5 ml of room-

temperature isopropanol, vortexed, and incubated for 2 minutes. Precipitated DNA was

loaded into a 30 ml syringe attached to a NucleoBond® finalizer and with minimal force

on the plunger the flow through was discarded. The precipitate was washed with 2 ml of

endotoxin-free 70% ethanol into the syringe and with minimal force on the plunger the

flow through was also discarded. The filter membrane was dried by pressing air through

the NucleoBond® finalizer while touching a tissue. The membrane-drying step was

127 repeated three times. The elution of the DNA was performed by the attaching of the

NucleoBond® finalizer into the outlet of a 1 ml syringe (provided) and adding 200 µl

nuclease free water. The water was pushed with minimal force through the NucleoBond®

finalizer and the DNA was collected in a sterile 1.5 ml microfuge tube. The concentration

of the DNA was determined with a ND-10000 Nanodrop (Fisher Scientific, San Diego,

CA).

Results

Formation of cJun constructs

The amplification of cJun fragments from RCASA plasmids was accomplished by

performing PCR (see materials and methods for detailed). All cJun genotypes including

the wild type cJun, cJun L40/42A, cJun R54A showed bands at 1kb, which is the

expected size product for cJun. The amplified cJun fragments were purified and the

concentrations were measured respectively for wild type cJun, cJun L40/42A, cJun R54A

as follow 35.8 ng/µl, 18.4 ng/µl and 44.4 ng/µl. Products were verified on an agarose gel

that showed the right expected size (Results not shown).

Purified cJun fragments were ligated into pGEM®-T Easy Vector, and the

transformation plates showed successful transformation with more than 150 white

colonies. The negative control did not have any growth (Results not shown). For the

verification of the cJun clones from the white colonies, five randomly picked colonies

from each of the cJun genotypes were tested by single colony PCR. The PCR products

showed the expected band at 1kb for all genotypes (Figure 1A,B). For each cJun

genotype, the plasmid from the bacterial culture that corresponded to positive PCR

128 products was purified. The concentrations were respectively for wild type cJun, cJun

L40/42A, cJun R54A as follow 235 ng/µl, 62.7 ng/µl and 273.8 ng/µl. The expected 1kb

fragments were observed from the restriction enzyme digestion of the purified clones

(Figure 1C,D). DNA sequencing results of the designed cJun constructs showed the right

frame of the GFP followed by the BAMH1 site and the inserted cJun (Figure 2). NCBI

BLAST analysis confirmed the cJun genotypes at the right orientation (Figure 3).

CJun fragments were cloned into the desired pLVX-EF1α-AcGFP1-C1 Vector starting with the digestion and purification of the vector (16.8 ng/µl). The digested vector resulted in a linear DNA product at the expected size 9.6 kb (Results not shown). CJun clones also were digested and purified, the purification concentrations for wild type cJun, cJun

L40/42A, cJun R54A as follow 8.8 ng/µl, 11.5 ng/µl and 6.6 ng/µl, respectively. The purified digested clones showed the expected bands at a 1kb (Result not shown). The LB plates for each of the transformation for the cJun genotypes showed more than 100 white colonies. The negative control showed 15 colonies. For each of the genotypes the 19 randomly picked colonies were purified and the concentrations were between 47 ng/µl and 130 ng/µl. All purified DNA was digested with BAMH1 and showed the expected band for cJun at 1kb (Figure 4). For each genotype, a second restriction digest was performed and one out of the 19 purified samples showed the expected size, which is a band at 1.6 kb for wild type cJun and cJun L40/42A and a band at 2.1 kb for cJun R54A genotype (Results not shown). DNA sequencing results of the designed cJun constructs showed the right frame of the GFP followed by the BAMH1 site and the inserted cJun

(Figure 5). NCBI BLAST analysis confirmed the cJun genotypes at the right orientation

129 (Figure 6). The concentration of the amplified verified cJun clones for wild type cJun,

cJun L40/42A, cJun R54A were as follows 186 ng/µl, 220 ng/µl and 318 ng/µl,

respectively. L40/42A genotype clone in protein database showing the mutation from

Lucine to Alanine at 40/42 sites.

The expression of the cJun clones was successfully observed in the transient

transfected 293T cells by western blot and fluorescence microscope. Western blot

analysis showed a band at 65 and 70 kDa that is the expected size for the fusion cJun.

The two observed bands are for different ATG in the cJun start site. Expected bands were

observed in all samples that got transfected with cJun clones. No bands were observed in

the negative control and sample that had the addition of pLVX-F1α-AcGFP1-C1 empty vector (Figure 7). GFP green signal were observed in all transient transfected 293T cells by fluorescence microscope.

Making stable cell lines

Two different vectors that consisted of cJun clones were used including pLVX-

EF1a-AcGFP1-C1 or the pLVX-Puro vector (For details see the main documents page

58-59). To make the cell lines, 293T cells were successfully transfected by cJun clones.

Fluorescence microscopy confirmed the presence of GFP signal that indicate the

successful transfection (Figure 8). The GoStix test (Clontech, Mountain View, CA) also

confirmed the indication of the viral in the 293T supernatant. The presence of two red

bands indicate the presence of the viral at >5 x 105 IFU/ml. Negative control showed

only one band indicating the absence of the viral in the supernatant of the negative

control (Figure 9).

130 The harvested viruses that included cJun clones were transduced into mESCs.

Examination by fluorescence microscopy showed low GFP expression 7 days post

puromycin selection in the transduced stem cells with either pLVX-EF1a-AcGFP1-C1 or pLVX-Puro that had cJun clones (Figure 10). Western blot analysis showed only one band at 40 kDa indicating the presence of endogenous cJun and the absence of the transfected clones (Figure 11). PCR and qPCR data also confirmed the absence of the transduced constructs (Figure 12,13).

131

Figure 1. Gel electrophoresis examined the presence of cJun genotypes in pGEM®-T Easy Vector by single colony PCR and enzyme digestion. A) Lane 2: single colony PCR of cJun L40/42A colony showed a band at 1000bp. B) Lane 2-6: single colony PCR of wild type cJun colonies; Lane 8-12: single colony PCR of cJun R54A colonies. All lanes showed bands at 1000bp. C) Lane 2-3: BAMH1 restriction enzyme digestion of cJun L40/42A genotype showed bands at 1000bp. D) BAMH1 restriction enzyme digestion of wild type cJun and cJun R54A genotypes showed bands at 1000bp. Lane 1: 1kb molecular weight standard. The gel electrophoresis was run in 0.7% agarose at 110 volts for 1 hour.

132

Figure 2. DNA sequences for the disigned cJun vectors from pGEM®-T Easy Vector clones showing the inserted cJun in frame with the GFP sequence. A- wild type cJun clone . B- cJun L40/42A clone. C- cJun R54 clone. Green= GFP sequence in the EF-vector. Red= BAMH1 site in the EF vector. Yellew= The start site of the inserted cJun formes.

133

Figure 3. BLASTX analysis data confirming correct mutations of cJun clones in pGEM®-T Easy Vector. A- Alignment of wild type cJun clone in protein database showing 100% identity. B- Alignment of cJun L40/42A genotype clone in protein database showing the mutation from Lucine to Alanine at 40/42 sites. C- Alignment of cJun R54A genotype clone in protein database showing the mutation from Argnine to Alanine at 54 site.

134

Figure 4. Gel electrophoresis examined the presence of cJun genotypes in pLVX-EF1α- AcGFP1-C1 Vector by BAMH1 enzyme digestion. Lane2: wild type cJun. Lane3: cJun L40/42A genotype. Lane4: cJun R54A genotype. All three lanes showed a band at 1000bp. Lane 1: 1kb molecular weight standard. The gel electrophoresis was run in 0.7% agarose at 110 volts for 1 hour.

135

Figure 5. DNA sequences for the designed cJun vectors from pLVX-EF1α-AcGFP1-C1 clones showing the inserted cJun in frame with the GFP sequence. A- wild type cJun clone . B- cJun L40/42A clone. C- cJun R54A clone. Green= GFP sequence in the EF-vector. Red= BAMH1 site in the EF vector. Yellew= The start site of the inserted cJun formes.

136

Figure 6. BLASTX analysis data showing the right mutation sites of cJun clones pLVX- EF1α-AcGFP1-C1 Vector. A- Alignment of wild type cJun clone in protein database showing 100% identity . B- Alignment of cJun R54A genotype clone in protein database showing the mutation from Argnine to Alanine at 54 site. C- Alignment of cJun

137

Figure 7. Western blot analysis indicating the presence of the cJun protein that induced from the transfected cJun vectors in 293t cells. Lane1, Fisher page ruler protein ladder. Lane 2, wild type cJun. Lane 3, cJun L40/42A. Lane 4, cJun R54A. Lane5, GFP. Lane 6, untreated 293t (control). All samples showed a band at 40kDa for endogenous cJun, samples in lane 2-4 showed bigger bands at 70kDa consisted of cJun protein that are GFP fusions. N=1.

138

Figure 8. Transfected 293T cells by cJun clones showing GFP signal. The presence of GFP expression in the images shows the successful transfection in 293T cells. 20X magnification. N=2. J1 cell line.

139

Figure 9. GoStix test indicating the presence of the virus in the media of the transfected 293T cells. The addition of 10ul of the supernatant resulting in two lines for positive virus production and one line indicating the absence of virus in the supernatant of the transfected 293T cells.1: Control (Mock). 2: cJun L40/42A genotype. 3: wild type cJun genotype. 4: cJun R54A genotype. 5: empty pLVX EF vector (positive control). N=2.

140

Figure 10. Images of transduced mouse embryonic stem cells showing low percentage of transduced cells. The images showed low GFP expression in the mESCs. The images were taken 7 days post puromycin selection. Images were taken at 20x magnification. N=2. J1 cell line.

141

Figure 11. Western blot of transduced mouse embryonic stem cells indicating the absence of the infused cJun protein. Lane1, Fisher page ruler protein ladder. Lane 2, GFP. Lane 3, cJun L40/42A. Lane 4, cJun R54A. Lane 5, wild type cJun. All samples showed a band at 40kDa for endogenous cJun and no presence of the transfected clones. The membrane had the addition of cJun antibody. N=1.

142

Figure 12. Gel electrophoresis showed the absence of YFP in the mouse embryonic stem cell lines after PCR amplification. Upper part: Lane 1, 1kb ladder. Lane 2, water. Lane 3- 4, negative control of untreated mESCs. Lane 6-7, mESCs transfected with cJun R54A. Lane 9-10, mESCs transfected with empty YFP vector. Down part: Lane 3-4, mESCs transfected with L40/42A.

143

Figure 13. The amplification of cJun in mouse embryonic stem cell lines by qPCR did not show an increase in the expression of cJun in the transduced sample. A- Lane 1, 100bp ladder. Lane 2, Water control. Lane 3, 1/10 dilution of cDNA from cJun L40/42A sample. Lane 4, 1/5 dilution of cDNA from cJun L40/42A sample. Lane 5, 1/2 dilution of cDNA from L40/42A sample. Lane 6, negative control cDNA. Lane 7, positive control of cJun plasmid. Lane 9, 1/10 dilution of cDNA from YFP. Lane 10, 1/5 dilution of cDNA from YFP. Lane 12, negative control cDNA. The results showed bands at 200bp for the cJun. B- CT value indicates higher gene expression in YFP sample in compare to cJun sample. N=1.

144 APPENDIX B.

Western and ICC Reagents

Table 1. Buffers components for Western blot application. Buffer Components Amount Company information

6x sample Sodium dodecyl 1.2g (Thermo fisher scientific, Waltham, MA) buffer sulfate Bromophenol 6mg (Sigma- Aldehyde®, Louis, MO) blue Glycerol 4.7ml (Sigma- Aldehyde®, Louis, MO) Tris 0.5M pH6.8 1.2ml (Thermo Fisher Scientific, Waltham, MA Deionized water 2.1ml 5x running Tris base 15.1g (Thermo Fisher Scientific, Waltham, buffer MA Glycine 94g (Thermo Fisher Scientific, Waltham, MA) Sodium dodecyl 50ml (Thermo fisher scientific, Waltham, MA) sulfate Deionized water 950ml Transfer 10x Tris-Gly buffer 100ml buffer Methanol 200ml (Thermo fisher scientific, Waltham, MA) Deionized water 700ml 10x Tris-Gly Tris base 58g (Thermo Fisher Scientific, Waltham, buffer MA Glycine 29g (Thermo fisher scientific, Waltham, MA) Deionized water 100ml 1x TBST Tris 12.11g (Thermo Fisher Scientific, Waltham, buffer MA NaCl 87.66g (Thermo fisher scientific, Waltham, MA) Tween-20 5ml (Thermo Fisher Scientific, Waltham, MA Deionized water 950ml Block 1x TBST buffer solution Bovine serum albumin 2% (Thermo fisher scientific, Waltham, MA) Nonfat dry milk 0.2% (Santa Cruz Biotechnology, Dallas, Texas) Deionized water

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Table 2. Primary and secondary antibodies for Western blot application. Antibodies that were used with mESCs and 293T cells. Primary Company and Working Incubation Incubation Antibodies catalog dilution time for time for 293T number mESCs samples samples cJun ( Abcam 1/1000 Overnight Three hours #32137) cJun phospho ( Abcam 1/1000 Overnight Overnight S63 #32385) cJun phospho ( Cell signaling 1/1000 Overnight Overnight S73 # 3270s) JNK ( Calbiochem # 1/1000 Overnight One hour 559304) phospho-JNK (Abcam # 1/1000 Overnight One hour ab124956) Oct4 ( Abcam # 1/1000 Overnight ab19857) Oct4A (Cell signaling 1/1000 Overnight # 2840s) GADPH (Cell signaling 1/1000 Overnight One hour #218S) Secondary Company and Working antibody catalog dilution number Goat anti- (Life 1/5000 45 minutes 45 minutes Rabbit IgG Technologies, (H+L) Grand Island, Secondary NY) Antibody

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Table 3. Primary and secondary antibodies for immunocytochemistry. Table consisted of antibodies that were used with mESCs and 293T cells. Primary Antibodies Company and Catalog Working number dilution cJun ( Abcam #32137) 1/250 cJun phospho S63 ( Abcam #32385) 1/200 cJun phospho S73 ( Cell signaling # 3270s) 1/200 JNK ( Calbiochem # 559304) 1/200 phospho-JNK (Abcam # ab124956) 1/200 Oct4 ( Abcam # ab19857) 5ug/ml Oct4A (Cell signaling # 2840s) 1/200 Nestin (StemCell Technologies # 1/100 01418) Anti-Insulin (Abcam #ab7842) 1/100 Monoclonal Mouse Anti- (Clone DM-1A # 691251) 1/100 Alpha Tubulin Secondary antibody Company and Catalog Working number dilution Texas Red Rabbit/IgG (Life Technologies, Grand 5ug/mL Island, NY) # A-6399 Texas Red mouse/IgG (Life Technologies, Grand 5ug/mL Island, NY) # T6390 Goat Anti-Guinea pig IgG (Abcam # ab6904-1) 1/200 H&L (FITC)