The Role of Fos and Junb in the Reprogramming of Acute Myeloid Leukemia Cells

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5-19-2019

The Role of Fos and JunB in the Reprogramming of Acute Myeloid Leukemia Cells

Kayla Bendinelli Dickinson College

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Recommended Citation Bendinelli, Kayla, "The Role of Fos and JunB in the Reprogramming of Acute Myeloid Leukemia Cells" (2019). Dickinson College Honors Theses. Paper 321.

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The role of Fos and JunB in the reprogramming of Acute

Myeloid Leukemia Cells

Kayla Bendinelli

Submitted in partial fulfillment of the Biochemistry and Molecular Biology Honors Requirement

Dr. Michael Roberts, Advisor, Committee Chair

Dr. Rebecca Connor, Reader

Dr. Dana Somers, Reader

May 8, 2019

The role of Fos and JunB in the reprogramming of Acute Myeloid Leukemia cells

Acute Myeloid Leukemia (AML) is the most common form of leukemia in adults and while it has a high remission rate, relapse with therapy resistance is common, indicating the need for more targeted and effective therapies. It is possible to reprogram AML cells in culture to undergo cell cycle arrest, differentiation into “normal” macrophage-like cells, and apoptosis using phorbol 12-myristate 13-acetate (PMA), a diacyl glycerol (DAG) mimic. While this is effective in “curing” leukemia in culture, PMA is too toxic to serve as a therapy in AML patients. During these PMA-induced changes, approximately 1250 genes change in expression.

The goal of this study was to see if the genes Fos and JunB, which are highly upregulated post

PMA treatment, are responsible for portions of the genetic reprogramming mediating these phenotypic changes. These genes are transcription factors and members of the AP-1 complex, which is known to play a role in regulating the cell cycle, differentiation and programmed cell death. In this study we show that Fos and JunB are capable of initiating specific responses by overexpressing them individually or together via transfection in the AML cell line, HL-60.

Title 1 Abstract 2 Table of Contents 3 Chapter 1: Introduction Acute Myeloid Leukemia 4 Activator Protein 1 (AP-1) 5 Introduction to AP-1 5 Fos Family 7 JunB 8 Role of AP-1 on morphological processes 9 Cell cycle arrest 8 Differentiation 10 Apoptosis 12 Clinical Significance 13 Summary 13 Chapter 2: The role of Fos and JunB in the Reprogramming of Acute Myeloid Leukemia Cells Abstract 15 Chapter Introduction Results Overexpression Validation 16 Target Gene Investigation 17 Cell Cycle Gene Expression 19 Assay 20 Macrophage Differentiation Gene Expression 20 Microscopy 20 Apoptosis 21 Summary 22 Chapter 3: General Discussion and Conclusion Discussion 23 Conclusion 25 Chapter 4: Tables and Figures 26 Chapter 5: Materials and Methods 50 Chapter 6: References 52

Chapter 1: Introduction

Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common form of adult acute leukemia with around 20,000 new cases a year1. Over the years the incidence of AML has been increasing while the mortality rate has remained relatively constant leading to the necessity for more effective therapies (Figure 1). AML is a high mortality disease, curable in 35-40% of patients younger than 60 years of age but only between 5%-15% of patients over the age of 60 2. While incidence of AML is higher in patients with a hematological disorder or who have received previous treatment including radiation for another cancer there is still no definitive cause for leukemia1. Most AML cases occur as a primary disease characterized by the proliferation of undifferentiated myeloid cells.

Cases of AML are categorized into subtypes based on the morphological and genetic mutations present in the patient’s leukemic cells. The most well-established example of a primary mutation driving oncogenic mutation is the translocation of chromosomes 15 and 17 which effects the retinoic acid receptor gene and causes the development of acute promyelocytic leukemia (APL) 1. In patients there are mutational changes to the genome resulting in activation of cell proliferation and inhibition of differentiation1. The types and number of mutations present in the cancerous cells contributes to the prognosis of the patient (Table 1). As mutations accumulate in the affected myeloid stem cells a main clonal population will develop with at least one subclonal population resulting in tumor heterogeneity2. While the mutated cells continuously divide there is a loss of myeloid lineage cells, including those involved with the immune response, and the crowding out of other necessary cells including red blood cells in the 5 bloodstream. This leads to the symptoms experienced by patients including fatigue, shortness of breath and easy bruising.

Once the genetics of a patient’s AML is determined, their course of treatment is decided.

Most will undergo induction therapy which consists of infusions of the chemotherapy agents cytarabine, which inhibits DNA synthesis, and anthracycline, which forms DNA intercalations, with the goal of achieving a complete response1. Trials are being undertaken to see if high doses of daunorubicin, which also forms DNA intercalations, will have similar success but using current therapies complete remission can be achieved in 60-85% of patients under 60 years of age2. Post-remission consolidation therapy consisting of chemotherapy and potentially bone marrow cell transplantation is required to eliminate residual disease and subclonal populations that may have escaped the initial treatment2. These are high risk and toxic therapies, especially for patients over the age of 60, so more targeted therapies are needed to produce durable responses. Therapies being tested include monoclonal antibodies that work to direct chemotherapy directly to the cancerous cells and inhibitors of the Signal transducer and activator of transcription 3 (STAT3) transcription factor which is activated in many cancer patients1.

These new targeted therapies focus on the genetic mutations of the cancer in hopes of selectively killing the cancer cells and sparing the patient from unnecessary side effects due to normal cell death.

Activator Protein 1

Activating protein-1 (AP-1) is a transcription factor composed of a dimer complex made up of members of the Fos, Jun, ATF and Maf families and has been linked to cell proliferation, differentiation and apoptosis3. These components are typically regulated as “immediate-early” 6 response genes which are a set of genes activated by present transcription factors in response to extracellular stimuli including growth factors and stress signals4. Some members of the AP-1 complex such as the Jun family proteins are capable of forming homodimers or heterodimers, while other members such as the Fos family only form heterodimers with other AP-1 members.

All of the AP-1 proteins contain a basic leucine zipper domain (bZIP) that allows the proteins to dimerize which aids in the stability of the protein5. Next to the dimerization domain the Fos and

Jun proteins contain a basic DNA binding domain that can bind to a palindromic motif with the sequence 5’-TGAG/CTCA-3” (Figure 2)3,6.

The individual members of the AP-1 family are regulated independently but once translated into protein they can form dimeric complexes that can activate downstream target genes that contain TPA-responsive elements (TREs), the AP-1 binding site5.Each AP-1 member’s binding domain has a different binding specificity for the target site allowing heterodimers to bind to a more diverse set of half-sites in different orientations than homodimers.

Target genes contain a version of the conserved consensus sequence for AP-1 in their promoter or enhancer regions that often contain some variation resulting in different binding capacities and thus outcomes for the varying Fos-Jun complexes7.

Under different conditions in different cell types, cells can contain a variety of AP-1 complexes, which are dependent on the expression levels of the individual proteins that are subject to change from outside stimuli. A stimulus can lead to an increase or decrease in a particular AP-1 gene leading to changes in the AP-1 complexes present within a cell that determine different transcriptomes8,3,7. A study done with mammary adenocarcinoma cell lines found that Fos was highly expressed in the non-invasive cells while in metastatic cells from the same tumor Fos was undetectable and Fra-1 and Fra-2 were strongly expressed8. A shift in AP-1 7 composition could be responsible for the progression of the tumor, however, this is very cell type dependent. Alternatively, a shift in AP-1 composition could assist in the reversion of the disease state. A study done on cervical cancer showed that Fos expression was high in the malignant cells and that treatment with the antioxidant pyrroline dithiocarbamate (PDTC) led to a reduction in Fos expression and an increase in c-Jun and Fra-18.

Fos Family

The Fos family consists of c-Fos, FosB, Fra-1 and Fra-2. These genes encode proteins with similar domains but can elicit different transcriptional responses, often depending on cell type. For example, Fos and Fra-1 can have similar effects including increased osteoblast differentiation and expression of genes involved with mammary tumor metastasis8,9. They can also show opposing functions as is the case in cervical cancer where Fra-1 is tumor suppressive and Fos tumorigenic8.The role of each of these genes is dependent on the cell type and the components of the AP-1 complex.

Fos, is an important member of the Fos family and is responsive to a variety of signals.

The promoter contains a cAMP-response element (CRE) as well as a serum-response element

(SRE), allowing Fos expression to respond to internal changes, changes in Ca2+ and cAMP, as well as external changes including changes in growth factors and cytokines4. Fos can also be induced by the Mitogen Activated Protein Kinase (MAPK) cascade, which plays a role in cell growth10.

Fos is involved in cell division in several ways. Fos has been shown to be oncogenic in certain conditions, but tumor suppressive in others. Fos’s role in promoting proliferation allows it to be oncogenic when complexed with c-Jun in chicken embryo fibroblasts10. However, Fos- 8

JunB dimers tend to have an opposing effect to Fos-c-Jun dimers and lead to an upregulation of tumor suppressive genes10. Clinically, Fos expression has been linked with both favorable and poor prognoses depending on tumor type. While high expression of Fos is associated with a poor prognosis in osteosarcomas and ovarian carcinomas, Fos expression has also been shown clinically to be higher in normal lung tissue compared to malignant tissues; this finding is, however, controversial8,9.

Jun Family

The Jun family consists of JunB, c-Jun and JunD. While c-Jun is a known oncogene,

JunB has been shown to have tumor suppressive characteristics in several cell types11. Similar to

Fos, the Jun gene transcription is responsive to external stimuli. Signals from cytokines and growth factors can initiate the mitogen-activated protein kinase cascade (MAPK), which includes the Jun N-terminal kinase that phosphorylates the Jun transactivation domain leading to its transcription and thus activation5.

Jun proteins can form homodimers or heterodimers with other members of the AP-1 complex, especially Fos proteins, through their c-terminal leucine domain. Of the Jun proteins c-

Jun is the most responsive to external stimuli, especially UV-radiation, and upregulates cell cycle genes including CyclinD and suppresses tumor suppressors contributing to transformation. On the other hand, JunB can have an inhibitory effect on active c-Jun due to possible dimerization leading to the opposite effect on cell cycle and tumor suppressive genes3,10.

Morphological Processes

The AP-1 complex is an essential transcription factor playing roles in the genetic programming of processes including development, growth and the stress response. This complex has been implicated in the induction and suppression of cell cycle arrest, differentiation and apoptosis depending on the dimer components, cell type and cell microenvironment9.

Cell Cycle

The cell cycle is the process by which cells replicate and consists of four phases, G1/G0,

S, G2 and M phase. In normal cells, a nondividing quiescent cell in G0, can receive signals from its environment that stimulate entry and progression through G112. During G1 the cell grows and prepares for DNA replication, if nutrients are insufficient the cell can revert back to G0. In most cancer cells it is the G0/G1 transition that is deregulated. At the end of the G1 phase there is a checkpoint, the restriction or R point, before S phase is initiated. During S phase DNA is replicated before entrance into G2 which consists of more growth and preparation for mitotic division. The final phase, M, is where mitosis occurs13. In hematopoietic progenitor cells of the myeloid lineage, cells can either differentiate into the various myeloid cell types or retain stem cell like properties and undergo controlled self-renewal13.

Each phase of the cell cycle contains a critical cyclin dependent kinase (CDK) that binds with a specific cyclin to form a cyclin-CDK complex that drives completion of that stage12.

Regulation of the cyclins is critical for regulated cell proliferation and there are two classes of cyclin-CDK inhibitors that can prevent formation and function of the cyclin-CDK complexes

(Figure 3)12,14. In AML the tight regulation of the cell cycle becomes deregulated resulting in uncontrolled growth. Flt3 mutations are common in many patients and result in the upregulation of the Phosphoinositide 3-kinase/Protien kinase B (PI3K/AKT) and MAPK pathways resulting in 10 the upregulation of cell cycle genes including CDK’s and increased cell cycle entry13.

Downstream of these pathways include genes like Fos and JunB which have both been implicated in the control of cell proliferation and growth.

Whether these genes play a role in cell proliferation or senescence is dependent on the cell type and dimer formed. In Fos-Jun dimers the deciding factor in whether the dimer will inhibit or promote proliferation is the Jun homologue. While c-Jun tends to be a proponent of proliferation, JunB and JunD reduce proliferation11. JunB is a critical downstream target of PU.1, a key transcription factor in myelomonocytic differentiation. Knockdown of PU.1 is sufficient to cause leukemia development in mouse models, and the reintroduction of JunB is capable of blocking AML cell proliferation15. Other studies have shown that a loss of JunB alone in the myeloid lineage cells is sufficient to cause leukemia development in mice models whom had increased proliferation of progenitor cells11,16.

JunB has a clear role in proliferation due to intracellular interactions. JunB is not only downstream of the growth inhibitor TGFβ but also has an effect on cell cycle regulators. It has been shown that JunB is a repressor of Cyclin D1, a key gene in cell cycle progression through the G1 phase, and an enhancer of the Cyclin D1-CDK inhibitor CDKN2A 3,15.

In addition to JunB, Fos has been linked to S-phase entry in osteoblasts as well as the expression of cell cycle promoters Cyclin e and B1, CDK2 and CDk4. However, Fos continues to be a paradox since it has also been linked with the expression of cell cycle inhibitors including

CDKN1A and CDKN2A8.

Differentiation

Differentiation is the genetic program by which a cell undergoes phenotypic changes to become a specialized cell type; for cells of the hematopoietic system this process is referred to as 11 hematopoiesis. This process begins with hematopoietic stem cells (HSC’s) which reside in the bone marrow and can divide indefinitely. HSC’s first give rise to progenitor cells which then give rise to more differentiated cells and so on, each step of the way the cells have a shorter renewal period and begin to take on more characteristics of their final differentiated state until they are fully differentiated and make up one of the many cells involved with the blood and immune systems 17,18. Progenitor cells play a key role in the differentiation of the hematopoietic cells into two branches. The Common Lymphoid Progenitor (CLP) gives rise to all lymphoid cells while the Common Myeloid Progenitor (CMP) gives rise to the megakaryocyte/erythrocyte lineage-restricted progenitor (MEP) and the granulocyte/macrophage lineage restricted progenitor (GMP). MEP differentiates into megakaryocytes and erythroid cells while GMP gives rise to macrophages and granulocytes (Figure 4) 17.

Many diseases that affect hematopoietic cells often involve the progenitor cells including leukemia. Cancer cells and HSC’s both share the capability of continuous division and several stemness pathways18. The conversion of a progenitor cell to a leukemic cell can lead to a loss of downstream differentiated cells. In acute promyelocytic leukemia (APL), a subtype of AML, there is a block in the promyelocytic stage leading to an increase in immature myeloid cells and a loss of differentiation17. One current therapy utilizes all-trans-retinoic-acid (ATRA) to force the differentiation of these cells without the toxicity of chemotherapy17.

Genes in the AP-1 family have been linked to the process of differentiation and hematopoiesis and may be able to force macrophage differentiation in our AML cells. Fos has been linked to the differentiation of osteoclasts due to activation of T-cells, which signal for the activation of nuclear factor κB and the Fos/AP-1 complex9. Osteoclasts are downstream of myeloid cells and share similar activation pathways. Fos has also been studied in macrophages 12 and was shown to be highly expressed in cultures of differentiated macrophages but not in their immature precursor cells indicating Fos expression may be linked with macrophage differentiation19. Another study done with myeloblastic leukemia M1 cells showed that overexpression of Fos genes lead to terminal differentiation and a decrease in leukemogenicity of the M1 cells when injected in nude mice20. Interestingly overexpression of JunB was able to induce similar changes in the M1 cells to a lesser extent, and was unable to fully compensate for a loss of Fos10.

JunB has been linked with myeloid differentiation due to its activation by the transcription factor Pu.1 which plays a key role in this process 15,21. JunB is involved with the signaling of the Notch and TGF-β pathways which lead to myeloid differentiation and a loss of

JunB results in a lack of differentiation and an increased number of progenitor cells in vivo as well22.

Apoptosis

DNA damage is “sensed” by cells allowing them to halt cell cycle progression and attempt to repair the damage. However, if the damage is too severe the cell will initiate programmed cell death, or apoptosis, in order to remove the cell with the corrupted and potentially harmful genome12. This is a process that is key in the removal of what could potentially develop into a cancerous cell and is evaded in cancer cells. Apoptosis consists of two sets of events. During the early events of apoptosis apoptotic signals result in the opening of channels in the mitochondrial membrane and the release of cytochrome c into the cytosol, which continues the cascade of events and activates caspase 9. This leads to the late stage events that consists of a cascade of one caspase cleaving and thus activating another caspase until the executioner caspases are 13 activated which degrade “death substrates” leading to the degradation of the cell12. In cancer cells the tight regulation of these events is lost allowing the cancer cells to evade apoptosis and to continue to thrive and divide.

Components of the AP-1 complex have also been shown to be involved with the apoptotic process in some cell types10. Fos has been shown to be involved with the induction of apoptosis in several cell types including lymphoid cells, embryonic Syrian hamster cell lines and myeloid leukemia cells16. This induction may be through the transcriptional regulation of Myc5.

Once again, the role of Fos proves to be cell dependent since it has been linked with anti- apoptotic effects in neuronal cell death.

JunB can also be pro-apoptotic. When JunB is inactivated in myeloid cells there is an upregulation of the anti-apoptotic genes Bcl2 and Bclxl which work to keep the channels that release cytochrome c into the cytosol shut and reduce apoptosis12,16. However, members of the

Jun family have been shown to inhibit p53 as well as preventing stress induced apoptosis6.

Clinical Significance

While these genes appear to play an important role in controlling cell growth, differentiation and apoptosis, which are the processes involved with the reprogramming of AML cells by PMA treatment, it is important to examine clinical correlations between survival and

AP-1 gene expression . Using The Cancer Genome Atlas (TCGA) AML database, it was found that Fos and JunB expression alone are not predictors of patient’s survival outcome27. When a metric was created that combined Fos and JunB expression it was found that there was still no statistically significant difference in survival (Figure 5). However, high expression of Fos and

JunB separately and together all trend towards a better survival rate which could reach 14 significance with more patient data. These data support the idea that the Fos-JunB dimer version of AP-1 may be important in controlling the genetic program of AML cells.

Summary

Three key morphological processes involved in the development of cancer and the reversal of the AML phenotype by PMA treatment are cell cycle regulation, differentiation and apoptosis. A key transcription factor in these processes is AP-1, which consists of several family members including Fos and JunB which have been shown to both promote and inhibit all three processes depending on the dimer formed and the cell type. During the PMA response Fos and

JunB are highly upregulated and may play a key role in the reprogramming of AML cells prompting the need for further investigation into the role of these genes in myeloid cells.

Chapter 2: The role of Fos and JunB in reprogramming Acute Myeloid Leukemia cells

Abstract

Determining which of the 1250 genes changing in expression during the reprogramming of HL-60 cells in response to PMA treatment is responsible for causing cell cycle arrest, macrophage differentiation and apoptosis opens up the opportunity to discover potential drug targets that can serve as better therapeutics for AML. The AP-1 complex is involved in all three of these morphological changes and two of its constituents, Fos and JunB, are drastically upregulated during the PMA response. These genes can dimerize to form a transcription factor that is activated by various stimuli to activate pathways involved with all of these processes.

These genes were overexpressed via transfection individually and together to investigate their role in reprogramming HL-60 cells independent of the other 1248 genes. These preliminary findings demonstrate that Fos and JunB are capable of inducing cell cycle arrest and to a lesser extent macrophage differentiation and apoptosis in HL-60 cells.

Chapter Introduction

HL-60 cells when treated with PMA undergo a series of phenotypic changes that are directed by changes in the transcriptome (Figure 6). During this process the expression of 1250 genes changes, around 100 of which encode transcription factors. PMA does not serve as a viable therapy due to its high toxicity, however, the key genes in this reprogramming may serve as potential drug targets. The aim of this study was to determine if the AP-1 transcription factor, specifically the components Fos and JunB, are key in these processes. From microarray data it was determined that during PMA treatment Fos expression is rapidly upregulated and remains high throughout the time course and this expression has been validated using RT-qPCR and 16

Taqman probes (Figure 7, 8c). Fos is a member of the AP-1 complex and forms heterodimers with members of the Jun family, especially JunB, which also rapidly goes up early during PMA treatment (Figure 7, 8d). While other AP-1 components did not experience a drastic change in expression it is possible that the Fos-JunB dimer may be playing an important role (Figure 7).

This study aimed to elucidate the role of Fos and JunB in the reprogramming of AML cells alone without inducing the PMA response. Fos and JUNB were overexpressed individually and together and changes in morphology and the transcriptome were evaluated.

Results

Overexpression Validation

The HL-60 cell line was transiently transfected through nucleofection (Figure 9) with the

GenScript® GenEZ™ ORF cDNA Clones containing either Fos or JunB under the control of a strong viral promoter (Figure 10). The original experiment was done with Fos over a time course to determine maximal overexpression and RNA was isolated at 4, 6, and 9 hours. RT-qPCR revealed no observable difference between the 6 and 9 hour time points as both exhibited maximal expression with a 575 fold induction (Figure 11). This prompted the use of an 8 hour time point for use in further experiments. A triplicate experiment was performed for both Fos and JunB individually and overexpression was again validated through RT-qPCR. Fos had about a 5,000 fold induction (Figure 12a) and JunB around a 45 fold induction (Figure 12b), both of which were consistent in following experiments.

Expression of the induced genes was also validated at the protein level. The plasmids used came with a tag at the end and an antibody specific to the additional amino acids (FLAG- tag). The western blot verified the presence of the tagged Fos at 40 kDa in the Fos transfected 17

HL-60 cells and JunB at 36 kDa in the JunB transfected HL-60 cells compared to the vector transfected HL-60 cells (Figure 13)23,24.

These experiments demonstrate that our transfection method is successful in the induction of our overexpressed genes at both the mRNA and protein levels.

Target Gene Investigation

Being a transcription factor the overexpressed Fos protein is going to have an effect on downstream target genes. cDNA was synthesized from the RNA samples and real time - quantitative PCR (RT-qPCR) using Taqman probes was used to evaluate the expression of multiple genes.The first samples used were vector and Fos transfected cells 8 hours post transfection (Figure 14). It was found that genes involved with apoptosis, BCL2; differentiation,

CD44; cell cycle, CDKN1A; and gene regulation, Fos, JunB, EGR1, CTSL; were only upregulated about 1.5 fold in the Fos vs vector transfected cells (Figure 14). While 8 hours was enough time for the Fos plasmid to become integrated into the HL-60 cells genome and to become highly expressed it was not enough time for Fos to have a downstream effect on other genes.

Another Fos transfection was performed and RNA isolated at 12, 16, 20 and 24 hours later giving Fos more time to work in the cell. Fos overexpression was validated and target gene expression investigated using Taqman probes and RT-qPCR. Fos expression was up 1,500 to

5,000 fold throughout the later time course (Figure 15a). The two genes with the greatest increase in expression at 8 hours were CDKN1A and JunB so we chose these genes to be evaluated first in the later time course. CDKN1A reached its highest expression levels first at 12 18 hours with a 2.5 fold increase in expression while JunB peaked at 16 hours with a 3.3 fold increase in expression (Figure 15b). These are still only modest changes in gene expression.

Fos requires another AP-1 component for heterodimer formation and activity. In the HL-

60 cells Fos is essentially turned off making the induction of Fos lead to a very high fold change.

Given that JunB, believed to be Fos’s partner in the PMA response, does not increase significantly after the introduction of Fos, it is likely that the endogenous JunB is easily depleted leading to excess Fos and a lack of active dimers. To ensure there was enough JunB to dimerize with Fos, another round of transfections were done with a vector transfected set of samples, a

Fos transfected set, a JunB transfected, and a Fos and JunB co-transfected set. RNA from all four groups was isolated at 4, 8, 12, 16, 20, and 24 hours post transfection to have a complete time course for all three groups. RNA was converted to cDNA in the manner described above and

RT-qPCR was run to validate the overexpression and to look at the expression of target genes.

The highest peak in expression of Fos in the Fos transfected cells was at 4 hours with a

6,000 fold increase (Figure 16a) and in the co-transfected set Fos peaked at 8 hours with an increase of 10,000 fold (Figure 16c). JunB expression in the JunB transfected and the co- transfected both peaked at 4 hours with 32 (Figure 16b) and 28 (Figure 16d) fold increases respectively. Overexpression was similar between the Fos and JunB single and co-transfected cells.

While validating the overexpression of the single transfections the dimer compliment expression was also evaluated to see if the two genes might regulate one another. In the Fos transfected cells endogenous JunB was elevated about 1.5 fold throughout the time course with a peak at 8 hours of 2.5 fold (Figure 17). This is fairly consistent with previous data. Curiously in 19 the JunB transfected cells Fos expression briefly increased to 2 fold at 4 hours before being downregulated to 0.25 of the expression of Fos in the vector control samples (Figure 17).

Cell Cycle

Expression of Cell Cycle Genes

Given that Fos-JunB dimers have been implicated in the cell cycle, the expression of regulatory components of the cell cycle were evaluated in the Fos and JunB single and co- transfected samples at the 8, 12, and 16 hour time points. These time points were chosen because in the late time course peak expression of target genes was at 16 hours. CDKN1A is an inhibitor of cyclin-CDK complexes and plays a key role in preventing cell cycle progression (Genecards).

Fos and JunB alone lead to modest increases in CDKN1A expression of around 3 and 2 fold respectively compared to vector transfected cells (Figure 18a). Together in the co-transfected cells these genes lead to a greater increase of CDKN1A of about 6 fold (Figure 18a). There appears to be an additive effect on expression.

The expression of another cyclin-CDK inhibitor, CDKN2B, was also evaluated. In the

Fos transfected cells CDKN2B is downregulated at 4, 8, and 12 hours but had a 6 fold increase in expression at 16 hours compared to vector transfected cells (Figure 18b). In the JunB transfected cells CDKN2B expression was down to half of that of the vector transfected cells (Figure 18b).

The co-transfected cells had a two fold increase in CDKN2B expression at 4 and 8 hours and then dropped below vector transfected levels at the 12 and 16 hour time points (Figure 18b).

CDKN2B is a regulator of the cyclin D- CDK4/6 complex and given the inconclusive results for CDKN2B the levels of Cyclin D1 were then investigated. In the Fos and JunB individually transfected cells there was a very slight increase in Cyclin D to 1.4 and 1.5 fold that 20 of the vector transfected cells respectively (Figure 18c). The co-transfected cells, however, saw an increase in Cyclin D expression of 2 fold at 4 hours, 3.5 fold at 8 and 12 hours, and 4.6 fold at

16 hours (Figure 18c).

Cell Cycle Assay

To evaluate if cell cycle arrest is occuring at the physiological level, HL-60 cells were transfected with vector, Fos, JunB, or Fos and JunB plasmid before being fixed and stained according to the Muse Cell Cycle® Kit protocol at 12, 16, 24, and 36 hours. Samples were run three times through the Muse Cell Analyzer which calculated the percent of cells in G0/G1, S, and G2/M phases (Figure 19). Early in the Fos time course there appears to be a shift of a higher percent of cells in G0/G1 and less in the G2/M phases compared to the vector treated cells.

However, at 24 and 36 hours there appears to be no difference between Fos and vector transfected cells (Figure 19e). The JunB transfected cells have a clear shift of more cells to

G0/G1 and less in S phase compared to vector transfected cells at all time points (Figure 19e).

The co-transfected cells had a similar shift of cells to G0/G1 from S phase at 12 hours as JunB transfected cells did compared to vector transfected (Figure 19e). At 16, 24, and 36 hours the co- transfected cells had a dramatic shift in cell phases, nearly all the cells appear to be in the G0/G1 phase (Figure 19e). It appears that JunB is sufficient to cause cell cycle arrest in HL-60 cells but is more efficient when overexpressed with Fos.

Macrophage Differentiation

Gene Expression

In order to investigate if the transfected cells were undergoing differentiation into macrophage like cells RT-qPCR was run to evaluate if the expression of various macrophage 21 differentiation markers changed. Past experiments showed the greatest target gene expression changes at 16 hours so samples of vector, Fos, JunB and co-transfected cells at 16 hours were used. For the genes CCL3 (Figure 20a) and TGFꞵ (Figure 20b) Fos and JunB acted similarly to the cell cycle genes. They individually had minimal increases in CCL3 expression of 1.2 and 1.5 fold respectively and together were capable of inducing a greater increase of 8.5 fold. TGFꞵ had a less drastic difference, Fos led to a 2 fold increase, JunB a 1.8 fold increase and the co- transfected had a 2.5 fold increase. For the other genes TNF (Figure 20e), CCL2 (Figure 20f) and

IL6 (Figure 20d) Fos and JunB individually led to greater increases in gene expression than they did in the co-transfection. For TNF the single transfections had increases of about 2 fold while the co-transfection had a 1.5 fold increase in expression. For CCL2 it was a more dramatic difference, Fos had an increase of 7 fold, JunB an increase of 8 fold and the co-transfection had an increase of 3 fold. For Il6 Fos had an increase of 3 fold, JunB 5 fold and the co-transfection 2 fold. In addition, all three treatments appear to have a similar effect on IL-1ꞵ (Figure 20c) with an increase of about 2 fold.

Microscopy

Changes in morphology were also investigated by using the Olympus CKX53 microscope at 20x magnification to view the cells at 12, 24 and 36 hours post transfection with and without the various plasmids. There appears to be no change in morphology over this time course in any of the treatments (Figure 21).

Apoptosis

To investigate if these genes were inducing apoptosis when overexpressed in HL-60 cells cells were transfected with either vector, Fos, JunB or co-transfected with both and harvested 36 22 hours later and stained according to the Muse Mitopotential® Kit protocol and run three times through the Muse Cell Analyzer from Millipore. There was a significant increase in the percent of depolarized live cells for all three treatment groups with a p-value less than 0.01 (Figure 22).

Depolarization of the mitochondrial membrane is an early apoptotic event and leads to the accumulation of the Mitopotential Dye which is read by the machine. This increase in depolarized cells indicates that apoptosis has been initiated in an increasing percent of cells.

Summary

The overexpression of Fos alone did not seem to induce the phenotypic changes observed in the PMA response. Despite changes in cell cycle gene expression the cell cycle assay revealed no clear difference in the percent of cells in the G0/G1 phase when compared to the vector transfected. There were some modest changes in macrophage differentiation markers but no clear morphological changes. The apoptosis assay did reveal the induction of apoptosis. All of this changed when Fos was co-transfected with JunB. JunB alone is capable of inducing changes in both cell cycle genes and the percent of cells in G0/G1 as well as macrophage markers and the induction of apoptosis. The effect on all three was further amplified when co-transfected with

Fos. It appears that the Fos-JunB dimer may be tumor suppressive in myeloid cells.

Chapter 3: General Discussion and Conclusion

These findings indicate that the AP-1 complex, more specifically Fos and JunB, likely play an important role in the PMA response in HL-60 cells. Fos and JunB alone and together are capable of inducing similar effects on the HL-60 cells as PMA. In the co-transfected cells it is more likely that Fos-JunB dimers are forming in place of JunB homodimers since the heterodimer has stronger binding activity and is thus more stable 8,10.

A key step in the reprogramming of HL-60 cells to normal macrophages during the PMA response is cell cycle arrest which we believe to be induced by JunB overexpression with and without Fos overexpression. While Fos and JunB individually had a modest effect on several cell cycle regulators, together they were capable of causing a more dramatic effect. Ironically this same effect was observed with a cell cycle promoter, however, as long as the inhibitor of this promoter is present in the cell this promoter will be unable to act. It was expected that the heterodimer would lead to a more dramatic effect on the transcriptome since Fos needs to form a heterodimer with other AP-1 family members and in Fos transfected cells there is no significant change in JunB expression and the low levels of endogenous JunB becomes the limiting factor in the formation of this complex. Compared to JunB transfected cells it was also expected that the co-transfection would have a greater effect since a Fos-JunB heterodimer binds with greater specificity to the target sequence than a JunB homodimer does7.

These cell cycle findings are further supported by the cell cycle assay results showing a shift of JunB and co-transfected cells from the S phase to G0/G1. JunB is a known regulator of the cell cycle and has been shown to prevent cell proliferation in myeloid cells11,15,16 . JunB is more stable as a heterodimer than it is as a homodimer which could explain the increased shift of cells to G0/G1 seen in the co-transfection 8,10. 24

While these genes appear to participate in cell cycle arrest, it is less clear if they are inducing the expression of macrophage differentiation genes. For some macrophage differentiation markers Fos and JunB induced greater increases in expression while in others the co-transfection did. Since both genes are implicated in macrophage differentiation and Fos needs to heterodimerize and JunB is more stable as a heterodimer it was expected that the co- transfection would lead to a greater increase for all macrophage genes that changed in expression. It is possible that in the co-transfection the overexpressed Fos and JunB are competing with other proteins at the promoters resulting in no binding and thus no transcription.

Within the AP-1 binding domain of the promoter of downstream target genes there is some variation leading to different binding specificities for the different dimers. Other AP-1 members including Maf and Atf are present in the cells and it is possible these other AP-1 dimers could be competing with the overexpressed heterodimer. There could also be the activation of an AP-1 inhibitor, further investigations are needed. Some of these gene changes were minimal while others were quite significant, more tests are needed to definitively say if these changes in macrophage differentiation genes is leading to a physiological change.

The final phase of the reprogramming of AML cells is apoptosis which we believe to be induced by Fos and/or JunB. The apoptosis assay at 36 hours showed a highly significant increase in depolarized live cells in all three treatment groups. Depolarization of a cell is an early marker of apoptosis since mitochondrial membrane breakdown is an initial step in the apoptosis cascade so this increase is indicative of higher levels of induced apoptosis in the transfected cells12. These results are preliminary and more tests with more time points are required for a definitive answer. 25

It appears that a shift in the AP-1 complex present in the HL-60 cells can revert the cancer program (Figure 23). It is possible that cJun-cJun dimers are present in the HL-60 cells and that the introduction of JunB-JunB dimers by JunB transfection induces cell cycle arrest and differentiation while the introduction of Fos-JunB dimers assists in differentiation and apoptosis.

Not only is AP-1 composition important in the role the AP-1 complex will play in the AML cells but also its abundance, Fos expression is low in the HL-60 cells and its overexpression is capable of causing significant induction of apoptosis.

Conclusion Acute Myeloid Leukemia is a devastating disease with a high relapse and mortality rate begging the need for more effective and targeted therapies. It is possible to reprogram HL-60 cells to act like normal macrophages that undergo cell cycle arrest, differentiation and apoptosis with PMA. While not a potential therapy itself, the 1250 genes that change in expression in response to PMA may elucidate novel drug targets including the AP-1 complex.

Fos and JunB are members of the AP-1 complex that are implicated in the pathways responsible for all three phenotypic changes of the PMA response and this study shed some light on how these genes are active in myeloid cells. Overexpression of JunB alone and in the co- transfection with Fos is capable of inducing cell cycle arrest while all three gene up-regulations appear to induce apoptosis and potentially macrophage differentiation. Further validation of these assays is needed to definitively conclude significant phenotypic changes as well as RNA sequencing experiments to better elucidate the global transcriptome changes occurring between the single vs co-transfected cells, which will also validate any synergistic effects taking place.

These preliminary results do show that Fos and JunB alone are capable of contributing to the reprogramming of HL-60 cells. 26

Chapter 4: Tables and Figures

Table 1: Mutations and patient outcome. Table depicts which mutations lead to a more favorable or adverse outcome. (Adapted from: Schlenk, 2004, Ref. 25).

Figure 1: Incidence and mortality rates of AML. The number of new cases of AML has been increasing over several decades while the death rate has remained the same. This prompts the need for more effective therapeutics.

Figure 2: Fos and JunB dimerization. Fos and JunB share similarities in their structure, both contain a leucine zipper motif adjacent to a basic DNA binding domain (A). The leucine zipper motifs allow for dimerization and the DNA-binding regions can then bind to AP-1 bind sites leading to transcription of downstream targets (B). (Adapted from Murphy, 2013; Ref. 26)

Figure 3: Cell cycle phases and important cyclin CDK complexes. Throughout the cell cycle different cyclins are upregulated allowing the formation of cyclin CDK complexes which drive the cell through to the next phase (A). Cell undergoes four phases to divide, G1, S, G2 and M and if not dividing rests in G0 (B). (Taken from Weinberg, 2007; Ref. 35)

Figure 4: Hematopoiesis map. A map of the differentiation of a hematopoietic stem cell (HSC) into the main progenitor cells that branch into the myeloid and lymphoid lineages and their final differentiated states. The lightning bolt indicates the progenitor believed to be transformed in the development of Acute Myeloid Leukemia.

Figure 5: Kaplan Meier plots for patients with high and low Fos and or JunB expression. Data was taken from the TCGA database which contains the sequenced genome of patients with AML. The patients in the top and bottom 20th percentile of expression for Fos (A) and JunB (B) expression were compared and a Multivariant Cox regression analysis revealed no significant difference in survival. (Adapted from Oncolnc; Ref 27). When looked at combined expression patients with higher levels of Fos and JunB (blue) had significantly better survival than those with low expression levels (gold) (C) (Figure courtesy of Ashir Borah).

Figure 6: Phenotypic changes in HL-60 cells post PMA treatment. HL-60 cells were treated with PMA and electron micrograph photos confirmed cell cycle arrest at 12 hours, differentiation around 24 hours and apoptosis by 48 hours (Figure courtesy of Simmons and Henson, unpublished results).

Gene 0.5 hr 1.0 hr 3 hr 6.0 hr 12 hr 24 hr

FOS 2.22 2.5 2.91 1.33 1.97 2.47

FOSB 0.3 1.1 0.94 -0.78 0.86 0.69

JUNB 3.07 3.34 2.52 3.13 3.25 2.96

JUND 0.15 0.6 1.43 2.39 1.1 1.75

MAF 0.03 0.51 -0.18 0.2 0.19 -0.31

ATF1 0.29 -0.54 0.94 0.79 -0.36 0.81

ATF2 -0.18 0.1 0.2 -0.08 -0.54 -0.4

Figure 7: Heat map data of AP-1 gene expression post PMA treatment. Heat map showing how the expression levels of various AP-1 genes changed over the time course post PMA treatment form microarray data. Fos and JunB were rapidly upregulated and expression remained high throughout the time course.

Figure 9: Transfection method. An overview of transfection by nucleofection method by Lonza. Cells were prepared in a transfection solution with plasmids of interest before having a charge transferred to them. This charge poked holes in the membrane allowing the plasmids to enter during membrane repair.

Figure 10: GenScript EZ Orf vector. A map of the GenScript® GenEZ™ expression vector containing a cloning site and the ampicillin resistant gene allowing for selection of transformed colonies (A). The gene of interest can be cloned into the cloning site under the CMV promoter which allows for high expression of the gene of interest in the transfected cells (B).

Figure 11: Fos overexpression time course in HL-60 cells. HL-60 cells were transfected with Fos plasmid from GenScript® GenEZ™ ORF cDNA Clones through nucleofection. RNA isolated at 4, 6 and 9 hours was converted to cDNA and RT-qPCR was done using a Fos Taqman Probe. Technical triplicates were run for each sample and ΔΔCT analysis revealed maximal expression at the 6 and 9 hours around 575 fold.

B. Figure 12: Fos and JunB overexpression in triplicate. HL-60 cells were transfected with Fos or JunB plasmid through nucleofection in triplicate. RNA samples were taken at 8 hours and converted to cDNA. RT-qPCR using a Fos (A) or JunB (B) Taqman probe was run to validate overexpression on all three samples and a ΔΔCT analysis was performed. Technical triplicates were performed on the biological replicates.

Figure 13: Western blot of tagged Fos and JunB validating expression at protein level. Protein was isolated from HL-60 cells transfected with 1) Vector, 2) Fos, 3) Vector, 4) JunB. Western blot was performed using the FLAG-tag anti-DYKDDDDK antibody and protein standards, on the left. Fos protein expression was confirmed at 40 kDa and JunB at 36 kDa.

Figure 14: Target gene expression in Fos transfected HL-60 cells. HL-60 cells were transfected with a plasmid containing Fos or just the vector and RNA was isolated 8 hours post transfection and converted to cDNA for RT-qPCR to look at target gene expression. Technical triplicates were done and experiments were later repeated. BCL2 (brown) is an apoptosis suppressor, CD44 (purple) is involved with hematopoiesis, EGR1 (yellow) is a transcription factor involved with cell survival proliferation and death, CTSL (orange) codes a protein that degrades other proteins, CDKN1A (grey) is involved with cell cycle regulation and JUNB (green) is part of the AP-1 complex and works with Fos to regulate genes involved with differentiation and apoptosis. There were minimal changes in target gene expression observed.

C. Figure 15: Fos overexpression in late time course. HL-60 cells were transiently transfected with Fos plasmid through nucleofection. RNA samples were taken at 12, 16, 20 and 24 hours post transfection and converted to cDNA. RT-qPCR using Taqman Probes was run to validate overexpression, technical triplicates were run for each sample and a ΔΔCT analysis performed Fos overexpression was first validated in the transfected cells (A). Probes for potential target genes CDKN1A (B) which is involved with cell cycle regulation and JUNB (C) which is part of the AP-1 complex and works with Fos to regulate genes involved with differentiation were run as well. Using the later time course resulted in greater changes in target genes than the 8 hour time point. 41

Figure 16: Fos and JunB overexpression validation in a full time course. HL-60 cells were transfected either with vector, Fos (A), JunB (B) or co-transfected with Fos and JunB ( C and D) plasmid and RNA was isolated at 4, 8, 12, 16, 20 and 24 hours and used to create cDNA for the purpose of running RT-qPCR using Taqman probes, technical triplicates and ΔΔCT analysis were used to look at gene expression. In the Fos transfected cells Fos expression peaked at 8 hours with a 6,000 fold increase (A). In the JunB transfected cells peak JunB expression occurred at 4 hours with a 32 fold increase (B). In the co-transfected cells Fos hit peak expression at 4 hours with 10,500 fold increase (C) and JunB hit peak expression at 4 hours with a 28 fold increase (D). Data was consistent with biological replicates.

Figure 17: Effects of Fos and JunB on each other’s native expression in transfected HL-60 cells. HL-60 cells were transfected either with vector, Fos plasmid (A) or JunB plasmid (B) and RNA isolated at 4, 8, 12, 16, 20 and 24 hours and used to create cDNA for use in RT-qPCR with Taqman probes and ΔΔCT analysis. Technical triplicates were used and data matches with subsequent experiments. In Fos transfected cells there was fairly consistent upregulation of JunB at around 1.5 fold with a peak at 2.5 fold at 8 hours (A). In JunB transfected cells Fos expression peaked at 4 hours at 2 fold before being downregulated below control levels (B).

C. Figure 18: Cell cycle regulators expression in Fos and/or JunB transfected HL-60 cells. HL- 60 cells were transfected either with vector, Fos, JunB or both plasmids and RNA was isolated at 8, 12, and 16 hours and used to create cDNA. RT-qPCR using Taqman probes for key cell cycle regulators and ΔΔCT analysis was performed. Technical triplicates were run per sample and experiments repeated with biological replicates. Fos and JunB alone lead to modest increases in CDKN1A, a cell cycle inhibitor, expression that increased over time but together had an additive effect on gene expression (A). Fos and JunB individually decreased CDKN2B, a cell cycle inhibitor, expression but FOS had a dramatic increase in expression levels at 16 hours. Meanwhile the co-transfection led to minimal changes in CDKN2B levels early on before also down regulating it (B). Fos and JunB individually had minimal upregulation of Cyclin D1, a cell cycle promoter, and together led to a steady increase in Cyclin D1 expression over time (C). 44

E. Figure 19: Cell cycle phases of vector transfected HL-60 cells. HL-60 cells were transfected with vector, Fos, JunB or Fos and JunB plasmid and fixed in 70% ethanol using the Muse Cell Cycle® Kit protocol at 12, 16, 24 and 36 hours post transfection. Fixed cells were later stained using the Muse Cell Cycle® Kit reagent and run through Muse Cell Analyzer from Millipore. The percent of cells in G0/G1 (blue), S (pink) and G2/M phases are shown (green). There is a clear increase of cells in G0/G1 in the JunB transfected cells and an even greater increase of G0/G1 cells in the co- transfected samples.

Figure 20: Expression of macrophage differentiation genes in Fos and or JunB transfected HL-60 cells. HL-60 cells were transfected either with vector, Fos, JunB or both plasmids and RNA was isolated at16 hours and used to create cDNA. RT-qPCR using a Taqman probes for macrophage differentiation genes and ΔΔCT analysis was run. Technical triplicates were used per sample and data matches subsequent experiments. It appears that overexpression of Fos and JunB alone led to modest changes while the co-transfection had a greater effect on some genes (A, B), while the single transfections had a greater effect on other genes than the co-transfection (D, E, F). For other genes all three treatments had a similar modest effect on gene expression (C) . Overall it appears there is some change in the expression of macrophage differentiation markers post overexpression of Fos with and without JunB.

Figure 21: HL-60 cells under microscope. HL-60 cells were either left untreated or transfected with vector, Fos, JunB or Fos and JunB plasmid before being viewed under a Olympus CKX53 microscope at 20x magnification at 12, 24 and 36 hours. There appears to be no morphological changes over this time course.

Figure 22: Apoptosis Induction in HL-60 cells post transfection. HL-60 cells were transfected with vector, Fos, JunB or Fos and JunB plasmid 36 hours before harvesting and staining with the Muse Mitopotential® Kit protocol. A t-test revealed that all three treatments had a significant increase in the percent of depolarized live cells indicative of apoptosis induction. *p-value<0.01.

Figure 23: Abundance and composition of AP-1 influences genetic program. The shift in AP-1 composition in HL-60 cells can induce cell cycle arrest, differentiation and apoptosis through the activation of various target genes.

Chapter 5: Materials and Methods Cell Culture HL-60 cells were purchased from ATCC and were kept at a concentration between

3x10^5 to 1x10^6 cells/mL. Culture conditions included being kept at 37℃ and 5% CO2. Cells were kept in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with penicillin, streptomyocin, amphotericin B (MP, Biomedicals 1674049) and 20% fetal bovine serum (ATCC).

PMA treatment HL-60 cells were treated with 16 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma; Phorbol 12-myristate 13-acetate, P1585) and RNA was isolated at specified times post treatment using RNeasy® Plus Mini Kit (QIAGEN; 74136).

Bacterial Transformation and Plasmid Preparation Competent E. coli were transformed with plasmids containing Fos (GenScript® NM_005252.3) or JunB (GenScript® NM_002229.2) under the control of the CMV promoter obtained from GenScript® GenEZ™ ORF. The E. Coli were transformed with the plasmid and the resulting colonies were inoculated in TB media containing Ampicillin the following day. Approximately 24 hours later plasmid DNA was isolated using the QIAprep® Spin Miniprep Kit (QIAGEN; 27104 and 27106). Concentrations and quality were checked using NanoDrop 2000 (Sigma; p5493-1L).

Transfection through Nucleofection Using the Amaxa® Cell Line 4D-Nucleotector® X Kit (V4XC-2024) 2*10^6 HL-60 cells/sample were transfected with 1.5µg Fos and/or JunB plasmid through Nucleofection®.

RNA Isolation The treated HL-60 cells were harvested at specified time points post transfection and the wells were washed with phosphate buffered saline solution (PBS) to ensure a maximum number of cells were isolated. RNA was isolated using the Quiagen RNeasy® Plus Mini Kit (QIAGEN; 74136). Concentrations and quality were checked using the NanoDrop 2000 (Sigma; p5493-1L). cDNA Synthesis cDNA was created from 1µg RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems; 4368814) and a GeneAmp PCR System 9700 (Applied Biosystems). The final cDNA was diluted to a volume of 60µl. cDNA was created for the purpose of RT-qPCR to view gene expression levels and due to low endogenous Fos expression a more concentrated cDNA was required to observe expression.

Real-Time Quantitative PCR (RT-qPCR) 50

RT-qPCR was performed using the QuantStudio 7 machine. Each reaction well contained 2µL cDNA, 10µL TaqMan® Gene Expression Master Mix (Applied Biosystems; 4369016), 1µL TaqMan probe and 7µL RNase free water. Every plate was run for 44 cycles using a standard run and ΔΔCT analysis was performed by the machine. Probes used: BCL2 (Applied Biosystems; Hs00608023_m1) CCL2 (Applied Biosystems; Hs00234140_m1) CD44 (Applied Biosystems; Hs01075864_1) CDKN1A (Applied Biosystems; Hs00355781_m1) CDKN2B (Applied Biosystems; Hs00793225_m1) CTSL (Applied Biosystems; Hs00964650_m1) CyclinD (Applied Biosystems; Hs01050839_m1) EGR1 (Applied Biosystems; Hs00152928_m1) FOS (Applied Biosystems; Hs04194186_s1) GATA2 (Applied Biosystems; Hs00231119_m1) IL1ꞵ (Applied Biosystems; Hs01555410_m1) IL6 (Applied Biosystems; Hs00985639_m1) JUNB (Applied Biosystems; Hs00357891_s1) RPS28 (Applied Biosystems; Hs02597258_g1)

Western Blots HL-60 cells were harvested and washed with phosphate buffered saline solution (PBS) 8 hours post transfection with either vector, Fos, or JunB plasmid. Cells were then lysed with 2X Laemmli Sample Buffer containing β-mercaptoethanol (Bio-Rad; 161-0737). The protein was then loaded onto a 10% SDS-PAGE gel containing a separating and a stacking layer. Once run the protein was transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Immobilon-P, PVDF; IPVH00010) and blocked in Tris-Buffered Saline with 0.1% Tween (TBST) and 5% non-fat milk. The membrane was then washed with TBST before being incubated with the anti-DYK (GenScript®; A00170-40) antibody for the tag engineered into the Fos and JunB plasmids for detection. This was followed by an incubation with the secondary antibody and washed again. The membrane was imaged using chemiluminescence (Thermo Scientific, PierceTM ECL Plus Western Blotting Substrate; 32132) and the FluorChem E imaging system (proteinsimple). In addition to the samples protein markers (BIO-RAD, Precision Plus ProteinTM Dual Color Standards; 161-0374) of known sizes were run to allow the determination of the size of present bands. Ꞵ-actin was also probed for to determine an equal amount of protein was loaded into all wells.

Muse Flow Analyzer Cell Cycle Assay 51

Post transfection HL-60 cells were harvested and the wells were rinsed with phosphate buffered saline solution (PBS). Cells were then washed with PBS and fixed in ice cold 70% ethanol following the Muse Cell Cycle® Kit protocol and stored at -20℃ (MCH100106). Cells were later stained with the Muse Cell Cycle® Kit reagent (MCH100106) and run through Muse Cell Analyzer from Millipore.

Muse Mitopotiential Assay HL-60 cells were transfected at 12, 16, 24 and 36 hours prior to isolation. The cells were harvested and the wells were rinsed with phosphate buffered saline solution (PBS) at the same time. Cells were then stained using the Muse Mitopotential® Kit protocol and run through the Muse Cell Analyzer from Millipore.

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