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Supplemental Figure 1 A Ptf1a-CreER; KrasG12D; Brg1f/f B Ptf1a-CreER; KrasG12D; Brg1f/f Brg1-positive PanIN Brg1-negative PanIN Brg1-positive PanIN Brg1-negative PanIN Alcian blue Alcian blue / / Brg1 Brg1 p16 p53 Supplemental Figure 1. The tumor suppressor genes p16 and p53 are down-regulated in BRG1-depleted PanINs. (A) Immunohistochemistry of serial sections for BRG1 and p16 in Ptf1a-CreER; KrasG12D; Brg1f/f mice. Arrowheads indicated p16-positive cells. Scale bars: 50μm. (B) Immunohistochemistry of serial sections for BRG1 and p53 in Ptf1a-CreER; KrasG12D; Brg1f/f mice. Scale bars: 50μm. A Supplemental Figure 2 ER Ptf1a Cre Ptf1a CreER Brg1 Brg1 Brg1 Brg1 G12D +Tam Brg1 Kras STOP Kras Kras KrasG12D R172H R172H p53 STOP Trp53 p53 Trp53 STOP d0 6m STOP Tam Sacrifice B R172H f/+ Brg1 ; Trp53 G12D ; Kras ER f/f Brg1 Ptf1a-Cre H&E Brg1 pERK Ki67 C d-14 d0 4m Tam Caerulein Sacrifice ; f/f G12D ;Brg1 ; Kras ER R172H Trp53 Ptf1a-Cre H&E Brg1 pERK Ki67 Supplemental Figure 2. Acinar-specific ablation of Brg1 attenuates PDA formation in the background of oncogenic KRAS and mutant p53. (A) The genetic strategy used to delete Brg1 and activate oncogenic Kras and mutant p53 in adult pancreatic acinar cells, in addition to experimental design for tamoxifen administration and analysis. (B) H&E staining and immunohistochemistry for BRG1, phospho-ERK and Ki67 of PDAs in Ptf1a-CreER; KrasG12D; Trp53R172H; Brg1f/f mice and littermate controls. Scale bars: 50μm. (C) Upper: Experimental design for tamoxifen administration, caerulein acute pancreatitis and analysis. Lower: H&E staining and immunohistochemistry for BRG1, phospho-ERK and Ki67 of PDAs in caerulein treated Ptf1a-CreER; KrasG12D; Trp53R172H; Brg1f/f mice. Scale bars: 50μm. Supplemental Figure 3 A B * ** 2 WT Ptf1a-Cre; 30 Brg1f/f 1.5 WT ****** * *** 20 1 f/f (% of ADM) +Tgf-α 10 0.5 0 0 Tgf-α - + - + Relative expression of mRNA Ptf1a-Cre; Brg1 WT Ptf1a-Cre; Brg1f/f acinar duct progenitor marker marker marker C D KrasG12D; Brg1f/f / KrasG12D 50 *** 2 40 Acvr2b Clu Rfx3 Sox4 Glis3 Foxo1 Ptpn6 Insr Invs Igf1r Bglap2 Il6ra Nkx2-2 Xbp1 Sox9 Ptf1a Eif2ak3 Selt Smad2 Kras 30 1 Pdx1 20 Pde3b Ad-Cre f/f 1/2 Onecut2 (% of ADM) 10 1/4 0 Kras; Brg1 Kras Kras; Brg1f/f Microarray mRNA fold change 1/8 Supplemental Figure 3. Primary acinar cell culture reveals that Brg1 promotes Sox9 expression. (A) Left: Brightfield images of acinar cell clusters from WT and Ptf1a-Cre; Brg1f/f mice after 5 days of culture in collagen gel with TGF-α. Scale bars: 50μm. Right: Quantification of cystic ductal strictures in 3D-cultured acinar cell clusters from WT and Ptf1a-Cre; Brg1f/f mice after 5 days of culture in collagen gel with or without TGF-α. n=3 per genotype; means ± SEM are shown. * P < 0.05, Student’s t test. (B) RT-PCR analysis of isolated acini from WT and Ptf1a-Cre; Brg1f/f mice. Genes important for pancreatic development and maintenance of pancreatic lineages are examined. n=3 per genotype; means ± SEM are shown. * P < 0.05, ** P < 0.01, and *** P < 0.001, Student’s t test. (C) Left: Brightfield images of acinar cell clusters from KrasG12D and KrasG12D; Brg1f/f mice after 5 days of culture in collagen gel. Cells were infected with Ad-Cre on day 0. Scale bars: 50μm. Right: Quantification of cystic ductal strictures in 3D-cultured acinar cell clusters from Ad-Cre-infected KrasG12D and KrasG12D; Brg1f/f acini after 5 days of culture in collagen gel. n=3 per genotype; means ± SEM are shown. *** P < 0.001, Student’s t test. (D) Fold changes in the expression of genes implicated in pancreas development and maintenance of pancreatic differentiation for Ad-Cre-infected KrasG12D; Brg1f/f acini after 24h of culture relative to control Ad-Cre-infected KrasG12D acini after 24h of culture. Supplemental Figure 4 A WT 1.6 Ptf1a-Cre; Brg1f/f 1.4 1.2 1 0.8 0.6 0.4 0.2 0 (Relative expression of mRNA) Supplemental Figure 4. Expression of upstream regulators of Sox9 in the pancreas is not significantly changed in Brg1-depleted acinar cells (A) RT-PCR analysis of Sox9 upstream regulators in isolated acini from WT and Ptf1a-Cre; Brg1f/f mice. n=3 per genotype; means ± SEM are shown. Student’s t test. Supplemental Figure 5 A Ptf1a-CreER; KrasG12D; Sox9OE; Brg1f/f Alcian blue / Brg1 Brg1-positive PanIN Brg1-negative PanIN B 185-fold change 267-fold change * 8 * 15 7 6 10 5 4 3 5 BRG1-positive BRG1-negative 2 1 Alcian blue positive area (%) 0 Alcian blue positive area (%) 0 Ptf1a-CreER; Ptf1a-CreER; Ptf1a-CreER; Ptf1a-CreER; Kras; Brg1f/f Kras; Sox9OE; Kras; Brg1f/f Kras; Sox9OE; Brg1f/f Brg1f/f Supplemental Figure 5. Sox9 overexpression accelerates KRAS-driven PanIN formation in mice. (A) Immunohistochemistry for BRG1 and alcian blue staining of Ptf1a-CreER; KrasG12D; Sox9OE; Brg1f/f mice. Representative BRG1-positive and BRG1-negative PanINs from the same sample are shown. Scale bars: 50μm. (B) Quantification of BRG1-positive and BRG1-negative PanINs in Ptf1a-CreER; KrasG12D; Brg1f/f mice and Ptf1a-CreER; KrasG12D; Sox9OE; Brg1f/f mice. n=3-4 mice per genotype; means ± SEM are shown. * P<0.05, Student’s t test. Supplemental Figure 6 A (8-16w old) day0 day3 Tam Sacrifice B Pdx1-FLP; FSFKrasG12D Pdx1-FLP; FSFKrasG12D; FSF-R26CAG-CreERT2; Brg1f/f 2.5 * 2 1.5 CC3 1 0.5 CC3 positive cells / PanIN 0 Pdx1-FLP; FSFKrasG12D Pdx1-FLP; FSFKrasG12D; FSF-R26CAG-CreERT2; Brg1f/f Supplemental Figure 6. Loss of BRG1 in established PanIN results in its apoptosis. (A) Experimental design for tamoxifen administration and analysis. (B) Immunohistochemistry for cleaved caspase 3 (CC3) in Pdx1-Flp; FSF-KrasG12D and Pdx1-Flp; FSF-KrasG12D; FSF-R26CAG-CreERT2; Brg1f/f mice 3 days after tamoxifen injection. Scale bars: 200μm. Right bar graphs are quantification for CC3 positive cells per PanIN lesion. n=3 per genotype; means ± SEM are shown. *P<0.05, Student’s t test. Supplemental Figure 7 A B PDA_2 PDA_3 PDA_1 PDA_2 PDA_3 PDA_4 PDA_5 ● ● ● ●● ●● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ●● ● ●● ● TP53 Nonsense mutation ● ● ●●● ● ● ● ●●● ● ● ● ● ●● ● ● ● ● ● ● ●●● ● ● ● ● ● ● ● ● ●● ● ● ● ●●● ● ● ●●● ● ●● ● ●●● ● ●●● ●● ● ●● ● ●●● ● ● ●● ● ● ● ● total CN KRAS Frameshift insertion/deletion total CN 0 2 4 CDKN2A Missense mutation 0 2 4 TGFBR2 Multiple mutation hetero hetero BRG1 Homozygous deletion SNPs SNPs Deletion ● ● ● ● ●● ●●● ● ● ●● ● ● ● ●● ●● ● ● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ●● ● ●● ● ● ●● ● ● ●● ● ● Loss of heterozygosity ● ● ● ●●● ●● ● ●● ●●● ● ● ● AsCN ● AsCN 0 1 2 0 1 2 C * 5000 ● 4000 3000 BRG1 Deletion Diploid 2000 Relative expression 1000 ● 0 Deletion Diploid BRG1 Supplemental Figure 7 Result of targeted sequence of BRG1-low human PDA samples (A) Mutational landscape of BRG1-low human PDA samples. KRAS, TP53, CDKN2A and TGFBR2 are shown as quality controls. Deletion of BRG1 is detected in 2 of 5 BRG1-low human PDA samples. (B) Detail of loss of chromosome 19p in PDA_2 and PDA_3. Total copy number (total CN) and allele specific copy number (AsCN) are shown. Copy number abnormalities are shown as black bar. Red triangle shows where BRG1 is located on. (C) Relationship between deletion of BRG1 and BRG1 mRNA expression from a cohort of 76 high purity samples in the TCGA data set. In the box-and-whisker plots, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers indicate minimum to maximum without outlier. *P<0.05, Mann-Whitney U test. Supplemental Figure 8 A B 8 9 SOX9 7.5 *** * 7 8 6.5 7 6 5.5 r=0.303 6 5 5 4.5 Relative mRNA expression 4 4 Relative mRNA expression of 5.5 6 6.5 7 7.5 Relative mRNA expression of BRG1 High Low High Low BRG1 BRG1 BRG1 BRG1 BRG1 SOX9 Supplemental Figure 8 BRG1 expression correlates with SOX9 expression in human PDAs. (A) Plots of mRNA expression of BRG1 and SOX9 from a cohort of 96 patients in QCMG data set. (B) The box-and-whisker plot demonstrates the differential expression for SOX9 between the BRG1-high (n =71, the higher 75%) and BRG1-low groups (n = 25, the lower 25%) from a cohort of 96 patients in the QCMG data set. In the box-and-whisker plots, horizontal bars indicate the medians, boxes indicate 25th to 75th percentiles, and whiskers indicate minimum to maximum without outlier. * P < 0.05 and *** P < 0.001, Student’s t test. Supplemental Table 1: Differently expressed genes in Kras G12D vs. Brg1 f/f acinar cell explants up down gene symbol fold change (log) gene symbol fold change (log) S100a6 3.7695728 G6pc2 -4.3786884 Krtap16-1 3.4862444 Fgl1 -3.9309574 Pecam1 3.46082 Lgals4 -3.1478798 Plvap 3.3369914 Ncor1 -3.1447362 Vaultrc5 2.9253636 Csrnp1 -3.12215 D8Ertd158e 2.8899298 Pdyn -3.1156846 Sprr1a 2.8063721 Espn -3.0293398 Cfb 2.7875106 Scg2 -2.9722324 Pxdn 2.7104712 Cyp2s1 -2.9414282 Zdhhc1 2.7012344 Slc39a10 -2.9306106 Ruvbl2 2.6697236 Zfp207 -2.9113906 Col1a1 2.549479 Rbm25 -2.8066914 Cxxc4 2.5407706 Hif3a -2.775671 Sprr2b 2.5220404 Ins1 -2.7639676 C5ar2 2.5090246 Ctsc -2.7582578 Parp3 2.5035262 Cib1 -2.7560214 Crhr1 2.4913382 Nnat -2.7315292 Tbc1d21 2.4566574 Fxyd3 -2.7256318 Oasl1 2.4398156 Nol3 -2.6992188 Sox15 2.411714 Kif5c -2.669723 Ecscr 2.3602772 Ins2 -2.6675257 Pglyrp1 2.3591118 Cyp2b10 -2.6431714 Vim 2.3278822 Crem -2.6197104 Atp1a3 2.317926 Hnrnpu -2.5793944 Rnase4 2.3134738 Smarca4 -2.5635176 Fhl2 2.3066534 Slco2b1 -2.5530882 Tiam1 2.303306 Espn -2.5276976 Fut4 2.302178 9530006C21Rik -2.5276084 Flna 2.2909526 Spock2 -2.5149002 Col4a2 2.283824 Gpr161 -2.507926 Cfb 2.246233 Rlf -2.5061864 Olfr700 2.237329 Ptprn -2.4990774 Sprr2k 2.2317066 Camta1 -2.4960784 Zmynd10 2.229248 Spaca6 -2.4613834 Ubash3b 2.21154 Oas1b -2.4521666 Stk10 2.1982666 Mrpl24 -2.4520208
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  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus

    A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus

    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
  • A Clinicopathological and Molecular Genetic Analysis of Low-Grade Glioma in Adults

    A Clinicopathological and Molecular Genetic Analysis of Low-Grade Glioma in Adults

    A CLINICOPATHOLOGICAL AND MOLECULAR GENETIC ANALYSIS OF LOW-GRADE GLIOMA IN ADULTS Presented by ANUSHREE SINGH MSc A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of Philosophy Brain Tumour Research Centre Research Institute in Healthcare Sciences Faculty of Science and Engineering University of Wolverhampton November 2014 i DECLARATION This work or any part thereof has not previously been presented in any form to the University or to any other body whether for the purposes of assessment, publication or for any other purpose (unless otherwise indicated). Save for any express acknowledgments, references and/or bibliographies cited in the work, I confirm that the intellectual content of the work is the result of my own efforts and of no other person. The right of Anushree Singh to be identified as author of this work is asserted in accordance with ss.77 and 78 of the Copyright, Designs and Patents Act 1988. At this date copyright is owned by the author. Signature: Anushree Date: 30th November 2014 ii ABSTRACT The aim of the study was to identify molecular markers that can determine progression of low grade glioma. This was done using various approaches such as IDH1 and IDH2 mutation analysis, MGMT methylation analysis, copy number analysis using array comparative genomic hybridisation and identification of differentially expressed miRNAs using miRNA microarray analysis. IDH1 mutation was present at a frequency of 71% in low grade glioma and was identified as an independent marker for improved OS in a multivariate analysis, which confirms the previous findings in low grade glioma studies.
  • Functional Screening of Alzheimer Risk Loci Identifies PTK2B

    Functional Screening of Alzheimer Risk Loci Identifies PTK2B

    OPEN Molecular Psychiatry (2017) 22, 874–883 www.nature.com/mp ORIGINAL ARTICLE Functional screening of Alzheimer risk loci identifies PTK2B as an in vivo modulator and early marker of Tau pathology P Dourlen1,2,3, FJ Fernandez-Gomez4,5,6, C Dupont1,2,3, B Grenier-Boley1,2,3, C Bellenguez1,2,3, H Obriot4,5,6, R Caillierez4,5,6, Y Sottejeau1,2,3, J Chapuis1,2,3, A Bretteville1,2,3, F Abdelfettah1,2,3, C Delay1,2,3, N Malmanche1,2,3, H Soininen7, M Hiltunen7,8, M-C Galas4,5,6, P Amouyel1,2,3,9, N Sergeant4,5,6, L Buée4,5,6, J-C Lambert1,2,3,11 and B Dermaut1,2,3,10,11 A recent genome-wide association meta-analysis for Alzheimer’s disease (AD) identified 19 risk loci (in addition to APOE) in which the functional genes are unknown. Using Drosophila, we screened 296 constructs targeting orthologs of 54 candidate risk genes within these loci for their ability to modify Tau neurotoxicity by quantifying the size of 46000 eyes. Besides Drosophila Amph (ortholog of BIN1), which we previously implicated in Tau pathology, we identified p130CAS (CASS4), Eph (EPHA1), Fak (PTK2B) and Rab3-GEF (MADD) as Tau toxicity modulators. Of these, the focal adhesion kinase Fak behaved as a strong Tau toxicity suppressor in both the eye and an independent focal adhesion-related wing blister assay. Accordingly, the human Tau and PTK2B proteins biochemically interacted in vitro and PTK2B co-localized with hyperphosphorylated and oligomeric Tau in progressive pathological stages in the brains of AD patients and transgenic Tau mice.
  • 4-6 Weeks Old Female C57BL/6 Mice Obtained from Jackson Labs Were Used for Cell Isolation

    4-6 Weeks Old Female C57BL/6 Mice Obtained from Jackson Labs Were Used for Cell Isolation

    Methods Mice: 4-6 weeks old female C57BL/6 mice obtained from Jackson labs were used for cell isolation. Female Foxp3-IRES-GFP reporter mice (1), backcrossed to B6/C57 background for 10 generations, were used for the isolation of naïve CD4 and naïve CD8 cells for the RNAseq experiments. The mice were housed in pathogen-free animal facility in the La Jolla Institute for Allergy and Immunology and were used according to protocols approved by the Institutional Animal Care and use Committee. Preparation of cells: Subsets of thymocytes were isolated by cell sorting as previously described (2), after cell surface staining using CD4 (GK1.5), CD8 (53-6.7), CD3ε (145- 2C11), CD24 (M1/69) (all from Biolegend). DP cells: CD4+CD8 int/hi; CD4 SP cells: CD4CD3 hi, CD24 int/lo; CD8 SP cells: CD8 int/hi CD4 CD3 hi, CD24 int/lo (Fig S2). Peripheral subsets were isolated after pooling spleen and lymph nodes. T cells were enriched by negative isolation using Dynabeads (Dynabeads untouched mouse T cells, 11413D, Invitrogen). After surface staining for CD4 (GK1.5), CD8 (53-6.7), CD62L (MEL-14), CD25 (PC61) and CD44 (IM7), naïve CD4+CD62L hiCD25-CD44lo and naïve CD8+CD62L hiCD25-CD44lo were obtained by sorting (BD FACS Aria). Additionally, for the RNAseq experiments, CD4 and CD8 naïve cells were isolated by sorting T cells from the Foxp3- IRES-GFP mice: CD4+CD62LhiCD25–CD44lo GFP(FOXP3)– and CD8+CD62LhiCD25– CD44lo GFP(FOXP3)– (antibodies were from Biolegend). In some cases, naïve CD4 cells were cultured in vitro under Th1 or Th2 polarizing conditions (3, 4).