Supplementary Figure 1
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1 Supplementary Figure 1 Control Pancreatitis
2 Supplementary Figure 2 A Panc Liver SI Spleen H 2 O B EZH2 fl/fl C EZH2 fl/fl 37bp EZH2 ERK2 D E 5 EZH2 fl/fl Fasting Glucose (mg/dl) EZH2 fl/fl
3 Supplementary Figure 3 A -EZH2 EZH2 fl/fl EZH2 ERK2 EZH2 fl/fl B EZH2 fl/fl Ctl D3 D9 Ctl D3 D9 EZH1 ERK2
4 Supplementary Figure 4 EZH2 fl/fl
5 Supplementary Figure 5 A Metaplastic Intermediates Ductal cells Acinar cells Dedifferentiation 1-2 days Proliferation 3-5 days Redifferentiation 5-7 days C B e; e; Cr SET H H2 p ZH2 EZ EZ E H2 EZ l fl/f 48- Day 3 EZH2fl/fl r -C SET l fl/fp48 2 Control Amylase Vinculin Day 1 EZH2 SET Control Trichrome Control E Day 3 Control EZH2 SET EZH2 SET EZH2fl/fl Day 3 EZH2fl/fl D -CD45 3 -CK19 6 p=.31 5 CK19 + cells/fov CD45 + cells/fov p=.15 EZH2fl/fl EZH2 SET EZH2fl/fl EZH2 SET
6 Supplementary Figure 6 Day 1 Day 5 EZH2 fl/fl
7 Supplementary Figure 7 A p48cre; EZH2 fl/fl Control Day 3 B EZH2 fl/fl PDX1/Ki-67 C Is PDX1/EZH2 Ki-67/DAPI
8 Supplementary Figure 8 A EZH2 fl/fl Control EZH2 fl/fl Cerulein Control Cerulein B EZH2 fl/fl p48cre; p48cre; ; p16 Ink4afl/fl Day 9 Day 3 Control Fraction Bound/Input IgG EZH2
9 Supplementary Figure 9 Kras G12D Kras G12D ; 1 month 3 month Pancreas weight (g) Kras G12D Kras G12D ; 1 month 3 month
10 Supplementary Figure 1 KrasG12D A KrasG12D; EZH2 SET 2 month 1 month Fraction of Total Ducts B KrasG12D KrasG12D;EZH2 SET N 1a 1b month N 1a 1b 2 month 2 3
11 Supplementary Figure 11 A B Kras G12D Kras G12D ; CD45 + Cells/2x FOV Kras G12D Kras G12D ; Side Scatter p48-cre Kras G12D Kras G12D ; % 2% 1% 16% 15% % 3% 2% 14% 16% % % % % % CD11c CD11b Gr-1 CD3 CD19 C Trichrome -SMA Kras G12D ; Kras G12D
12 Supplementary Figure 12 A PanIN/FOV Vehicle Nimesulide 2 Kras G12D Kras G12D ; B Vehicle Nimesulide Kras G12D
13 SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure 1: EZH2 upregulation in human pancreatitis samples. Histological sections from human control and pancreatitis samples (obtained from the Tissue Acquistion and Banking Services of the NYU Experimental Pathology Core Facilities) were stained with EZH2 antibody. Results are representative of the staining pattern seen in 3 pancreatic patients. Scale bar, 5. Supplementary Figure 2: EZH2 deletion in EZH2 ΔSET animals. (A) PCR analysis of DNA from abdominal organs of EZH2 ΔSET mice. The 37bp fragment corresponding to the recombined allele is present only in the pancreas. Panc, pancreas; SI, small intestine. (B) Western blot for EZH2 expression in pancreatic lysates of EZH2 fl/fl and EZH2 ΔSET. ERK2 serves as a loading control. (C) General appearance of pancreata from EZH2 fl/fl and EZH2 ΔSET mice. (D) H&E stained sections from EZH2 fl/fl and EZH2 ΔSET mice. Scale bar, 5. (E) Fasting glucose levels (means +/- SD) measured in EZH2 fl/fl and EZH2 ΔSET mice. Data in all panels are from 2-month-old mice and are representative of 3 mice per genotype. Supplementary Figure 3: EZH1 and EZH2 expression during pancreatic regeneration. (A) EZH2 expression was analyzed from EZH2 fl/fl and EZH2 ΔSET mice on day 3 after final cerulein injection by immunohistochemical staining of pancreata (left) and western blot of pancreatic lysates (right). Scale bar, 5. ERK2 serves as a loading control. (B) Western blot shows no compensatory EZH1 upregulation in EZH2 fl/fl or p48-
14 Cre;EZH2 ΔSET pancreata upon cerulein injection. ERK2 serves as loading control. Data in all panels are from 2-month-old mice and are representative of 3 mice per genotype. Supplementary Figure 4: Impaired regeneration of the exocrine pancreas in p48- Cre;EZH2 ΔSET mice. Representative images (n = 3 for each genotype) of H&E-stained sections from tissues harvested 21 days following final injection of cerulein. Scale bar, 5. Supplementary Figure 5: Pancreatic regeneration and loss of amylase during injury phase. (A) A schematic depiction of the key steps that mediate the process of pancreatic regeneration in response to injury. (B) Western blot analysis of amylase expression demonstrating an equivalent level of acinar cell loss in EZH2 fl/fl and EZH2 ΔSET mice on day 1 after final cerulein injection. Results are representative of data obtained from 3 animals per genotype. (C-E) Trichrome C staining (C), immunohistochemical staining for CD45 (D) and CK19 (E) from EZH2 fl/fl and EZH2 ΔSET pancreata on day 3 after final cerulein injection compared to control. For panel C, results are representative of data obtained from 3 animals per genotype. For panels D and E, quantifications were performed by counting CD45+ or CK19+ cells per four randomly selected 2X fields of view (FOV) (5 mice per genotype) on day 3 after final cerulein injection. Scale bars, 5 (C, D) and 2 (E).
15 Supplementary Figure 6: Acinar cell cultures from EZH2 fl/fl and EZH2 ΔSET pancreata. Acinar cells from EZH2 fl/fl and EZH2 ΔSET mice display similar morphologic transition at days 1 and 5 in culture. Scale bar, 5. Supplementary Figure 7: EZH2 is required for the proliferative expansion of the metaplastic epithelium during regeneration. (A) Immunofluorescent detection of Ki67- positive cells at days and 3 after the final injection of cerulein shows enhanced proliferation in EZH2 fl/fl but not EZH2 ΔSET pancreata. Scale bar, 5. (B) Double immunofluorescence staining for PDX1 and Ki67 at day 3 after final cerulein injection demonstrates that the majority of Ki67+ proliferating cells are confined to PDX1- expressing metaplastic lesions (marked by asterisks) in EZH2 fl/fl but not p48- Cre;EZH2 ΔSET pancreata. Scale bar, 2. (C) Double immunofluorescence for PDX1 and EZH2 on day 3 after final cerulein injection shows that EZH2 is expressed in PDX1- positive metaplastic epithelium (marked by asterisk) of the regenerating pancreas. Is, islet cells. Scale bar, 2. Images shown in all panels are representative of data obtained from 3 animals per genotype. Supplementary Figure 8: EZH2 controls pancreatic regeneration through the suppression of p16 Ink4a expression. (A) ChIP analysis from pancreata harvested at day 3 after final cerulein injection shows recruitment of EZH2 to the p16 Ink4a locus in EZH2 fl/fl but not EZH2 ΔSET pancreata. Results represent the means +/- SD of three independent determinations. (B) Pancreata from EZH2 fl/fl, EZH2 ΔSET and p48- Cre;EZH2 ΔSET ;p16 Ink4afl/fl mice were harvested on days 3 and 9 following final cerulein
16 injection. H&E analysis of the sections showed that impaired regeneration was rescued in EZH2 ΔSET ;p16 Ink4afl/fl mice. Images shown are representative of data obtained from 3 animals per genotype. Scale bar, 5. Supplementary Figure 9: General appearance and weight comparison of p48- Cre;Kras G12D and Kras G12D ;EZH2 ΔSET pancreata. Pancreata from p48- Cre;Kras G12D ;EZH2 ΔSET mice are enlarged at 1 month and smaller at 3 months, relative to pancreata from Kras G12D mice assessed by appearance and weight. Data are representative of 3 mice of each genotype and age. Supplementary Figure 1: EZH2 deficiency accelerates PanIN progression. (A) Lower magnification H&E stained sections from 1- and 2-month-old Kras G12D and p48- Cre;Kras G12D ;EZH2 ΔSET mice. Arrowheads indicate early PanIN lesions, and asterisks indicate advanced lesions. Scale bar, 1. (B) Histological progression of PanINs in Kras G12D and Kras G12D ;EZH2 ΔSET pancreata at indicated ages (N, Normal; 1, 1a, 1b, 2, 3, PanIN Stage). Results represent the means +/- SD of lesions per section identified at 1 month (5 mice per genotype) and 2 months of age (5 mice per genotype). At least 4 sections were analyzed per animal. () p<.5. Supplementary Figure 11: EZH2 deficiency results in an exacerbated stromal response. (A) Immunohistochemical detection of pan-leukocyte marker CD45 reveals enhanced leukocyte infiltration in the pancreas of Kras G12D ;EZH2 ΔSET relative to p48- Cre;Kras G12D pancreata. Quantification was performed by counting CD45+ cells in ten
17 randomly selected 2X FOV in 4 animals. Scale bar, 5. (B) Flow cytometry profiles of pancreata from mice of the indicated genotypes demonstrating enhanced recruitment of dendritic cells (CD11c), neutrophils (CD11b), and macrophages (Gr-1), but not T (CD3) or B (CD19) cells to the pancreas of Kras G12D ;EZH2 ΔSET mice. Results are representative of 4 separate experiments with 3 mice per genotype. (C) Enhanced collagen deposition (Trichrome stain, scale bar, 1 ) and desmoplasia [smooth muscle actin ( -SMA) immunohistochemistry; scale bar, 5 ] in Kras G12D ;EZH2 ΔSET pancreata at 2 months of age. Images shown are representative of data obtained from 3 animals per genotype. Supplementary Figure 12: Nimesulide treatment and PanIN progression in p48- Cre;Kras G12D versus Kras G12D ;EZH2 ΔSET pancreata. (A) PanIN lesions per ten randomly selected 2X FOVs were counted in H&E stained pancreatic sections (5 mice per genotype) from vehicle and nimesulide treated 1 month old Kras G12D and Kras G12D ;EZH2 ΔSET mice. (B) H&E images of pancreata from vehicle and nimesulide treated, 1 month old Kras G12D mice show that nimesulide treatment does not effect Kras G12D pancreata. Arrowheads indicate early PanIN lesions. Images shown are representative of data obtained from 3 animals per genotype. Scale bar, 5.
18 SUPPLEMENTARY MATERIALS AND METHODS Immunofluorescence and immunohistochemistry Immunofluorescence was performed on 8μm thick frozen sections of pancreas. Slides were air dried for 15min and fixed with ice cold 4% paraformaldehyde for 1min. Sections were permeabilized with.2% Triton-X1 and blocked with 1% chicken serum for 1hr. Primary antibodies were diluted in 1% chicken serum,.5% Tween 2 and incubated overnight at 4 o C. Secondary antibodies raised in chicken (Invitrogen) were incubated on sections for 1hr. Immunohistochemistry was done on 5 μm sections from paraffin embedded blocks. Sections were deparaffinized and rehydrated, followed by quenching in 3% H 2 O 2 in methanol. Antigen retrieval was performed in 95 o C.1M citrate buffer (ph 6) with.5% Tween 2. Sections were blocked with 5% goat serum. Primary and secondary antibodies were diluted in 1% BSA/PBS. After secondary antibody incubation VectaStain Elite ABC Reagent (Vector Laboratories) was applied and the resulting antibody/conjugate was developed with diaminobenzamidine (Sigma). Sections were then counterstained with hematoxylin and mounted with Permount (Fisher). Hematoxylin and eosin staining was also performed on paraffin sections. Tissue Staining Antibodies used are rabbit anti-ezh1 (Margueron et al. 28), rabbit anti-ezh2 (Kuzmichev et al. 24), rabbit anti-h3k27me3 (Millipore, 7-449), goat anti-pdx1 (C.V. Wright, Vanderbilt University), rabbit anti-ki67 (Novacastra), goat anti-amylase (Santa Cruz), rabbit anti-amylase (Sigma), rabbit anti-sma (Abcam), rabbit anti-
19 p16 INK4A (Santa Cruz), rat anti-ck19 (TROMA-III-c, developed by R. Kemler, obtained from Developmental Studies Hybridoma Bank under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242). Trichrome C staining was performed by the histopathology core at NYU School of Medicine. Quantitative immunofluorescence analysis for amylase Quantitative immunofluorescence analysis of amylase expression was done using ImageJ Vs The freehand outline tool was used to outline all areas staining for amylase in 8-1 random fields of view (4 2 /field of view) from the pancreata of two mice for each timepoint and condition. The measured area was used to determine a percentage of amylase positive cell staining / field of view. Protein and RNA extraction For proteins, pancreata were flash frozen and homogenized in lysis buffer containing 5mM Tris ph 7.4, 4mM NaCl,.5% NP-4, 5mM EDTA ph 8, and 5mM NaF with protease inhibitors (aprotinin, leupeptin, sodium orthovanadate, PMSF, DTT). For RNA, pancreata were flash frozen in liquid nitrogen and resuspended in pre-chilled RNAlater ICE (Ambion) to be incubated at least overnight at -2 o C. A small piece of frozen pancreas was ground with a mortar and pestle, and the RNEasy kit (Qiagen) was used to extract RNA from the ground tissue.
20 Quantitative PCR RNA was reverse-transcribed using Quantitect Reverse Transcription Kit (Qiagen). All primers were designed using Primer 3 software available from MIT. qpcr was performed using SYBR Green Master Mix (USB) and amplified using a Stratagene Mx35p. Results were analyzed using MxPRO software. Acinar cell isolation and culture Pancreata were harvested and acinar cells were isolated as described (Sawey et al. 27). Cells were plated and maintained on Matrigel using Waymouth media supplemented with 1% FBS, 4μg/mL dexamethasone,.4mg/ml trypsin inhibitor and penicillin/streptomycin. Chromatin immunoprecipitation Chromatin was isolated by weighing and mincing pancreatic tissue in PBS with protease inhibitors. Proteins were crosslinked with 1% formaldehyde for 15min at room temperature. Then glycine was added to a final concentration of.125m. Nuclei were then sonicated using a bioruptor for 15min twice to obtain chromatin. Crosslinked complexes were isolated by immunoprecipitation with the indicated antibodies. Crosslinking was reversed by incubation at 65 o C for 15min and 2μg RNase A and 4μg proteinase K were added to samples. DNA was isolated using phenol-chloroform extraction and ethanol precipitation and subjected to quantitative PCR.
21 Flow cytometry Pancreas was minced on ice with a razor blade and then digested with 1mg/ml collagenase for 1min at 37 o C. After lysing red blood cells, the sample was filtered through a 7μm mesh. Fc block was used before adding appropriate fluorescent conjugated antibodies. Fasting Glucose Measurements After overnight fasting, mice tails were cut and blood was applied to a Medisense Precision QID glucose monitor.
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