Generation of conditional acinar-specific loss of Arid1a Mice
Arid1afl/fl floxed mice have loxP sites Jackson Laboratory (Stock No: 027717) were bred to Ela1-Cre/ERT2 transgenic mice (Stock No: 008861) to generate Arid1afl/fl; Ela1-Cre/ERT2 mice. Ela1-Cre/ERT2 have a tamoxifen-inducible Cre-mediated recombination system driven by the rat elastase 1 pancreatic promoter. All mice were housed in pathogen-free animal facilities and handled in accordance with the guidelines of the Institutional Animal Care and Use Committee at Taizhou University Hospital. Cre recombinase activity was induced by daily intraperitoneal injection of tamoxifen (Sigma-Aldrich, St. Louis, MO) over five days. Arid1afl/fl; Ela1-Cre/ERT2 mice were administered tamoxifen at a dose of 0.25mg/g body weight or equivalent corn oil. Excision of Arid1a was evaluated in mice 1 week after injection by PCR of DNA collected from mouse tissues. Primers of PCR genotyping were listed in Supplementary Table 1.
Cerulein-induced acute pancreatitis (AP)
1 week after the mice were administrated with tamoxifen, acute pancreatitis (AP) was induced by intraperitoneal injections of cerulein (Sigma-Aldrich, Steinheim, Germany) eight times hourly for two consecutive days with a total of 0.1 µg/g body weight per mouse, as previously described [27]. The final day of injection was considered as day 0. Blood was collected at 1, 3, 5, 10 hours, 1, 5 and 7 days after the last cerulein injection to examine the serum amylase and lipase levels.
Tissue processing and histology
Mice were euthanized at indicated time points, pancreata were dissected in ice-cold PBS and separated into multiple fragments. Formalin-fixed, paraffin-embedded (FFPE) pancreas tissue was cut into 6-8 µm sections and stained with H&E. Pancreatic damage was semi-quantitatively assessed by scoring 3-5 random slides per mouse according to quantitative method described in the references [28-30]. Briefly, the semi-quantitative scoring system for edema and acinar necrosis were graded from 0 to 3 according to the following criteria: edema: 0, absent; 1, focally increased between lobules; 2, diffusely increased between lobules; and 3, acini disrupted and separated; and acinar necrosis: 0, absent; 1, periductal necrosis (5%); 2, focal necrosis (5-20%); and 3, diffuse parenchymal necrosis (20-50%). Total pancreatitis was calculated by grade of inflammation × distribution of inflammation. The scoring was performed in a blinded manner by an experienced pathologist.
Serological examination of amylase, lipase, and IL-6 cytokine
The serum activity of amylases and lipases were measured by enzyme dynamics chemistry using commercial kits, according to the manufacturer’s instructions in a Roche/Hitachi modular analytics system (Roche, Mannheim, Germany). Serum IL-6 level was determined by enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Quantikine; R&D Systems, Minneapoils, MN, USA).
Immunohistochemistry and immunofluorescence assay
FFPE samples were cut into 5µm sections. After deparaffinization and rehydration by passage through xylene and a graded alcohol series, heat-induced antigen retrieval was achieved by cooking in antigen unmasking solution (Vector Laboratories). Endogenous peroxidase activity was inactivated by 0.3% hydrogen peroxide treatment for 10 min at room temperature. After blocking with TBS containing 3% BSA, sections were incubated with the primary antibody at 4˚C overnight. For immunohistochemistry, sections were incubated in secondary antibody for 1 hour and then developed using Avidin-Biotin Complex (ABC) and Diaminobenzidine (DAB) kit (Vector Laboratories) and counterstained with hematoxylin. For immunofluorescence assay, slides were subject to primary and secondary antibody incubations, counterstained with DAPI (Sigma-Aldrich; St. Louis, MO).
Isolation and culture of primary acinar cells
Primary acinar cells were isolated from mice pancreas using a collagenase digestion as previously described procedure [31, 32]. Briefly, pancreata were immediately removed and rinsed twice with ice-cold Hank's Balanced Salt Solution (HBSS, Thermo Fisher Scientific). Pancreatic tissue was minced into 1-5 mm pieces and digested with collagenase P (Roche Applied Science, Mannheim, Germany) for 15 min at 37 ˚C. Isolated acini were washed three times in ice-cold HBSS containing 5% fetal bovine serum (FBS), and then filtered through 500 and 105-µm nylon meshes (Spectrum Laboratories, CA, USA). Acini were collected by centrifugation and re-suspend in 10 ml Waymouth medium (Invitrogen, Carlsbad, USA) containing 1% FBS, 0.1 mg/ml soybean trypsin inhibitor, 1 μg/ml dexamethasone. Culture dishes were coated with rat tail collagen type І (Sigma-Aldrich) in 0.02 N acetic acid, pH 3.67 solution for 1 hr at 37 ºC, and the isolated primary acini were seed into the prepared plates coated with matrix scaffolds in triplicates. Acinar cells were infected with lentivirus of interest and incubated for 3-5 hours before embedding in the collagen/waymouth media mixture. Numbers of ducts were counted under a microscope. Acinar explants were seeded in triplicates, and cells clusters were counted from at least 3 optical fields/well and reported as a percentage of acinar clusters and ring-like spheres.
RNA isolation and reverse transcription quantitative-PCR (RT-qPCR)
Total mRNA was isolated using Trizol reagent (Ambion) following manufacture’s instruction. 1 μg RNA was reverse transcribed using oligo dT primer was performed with iScriptTM gDNA Clear cDNA Synthesis Kit (BIO-RAD). Synthesized cDNAs were diluted 2-fold with water, and regions of interest were amplified by SYBR® Green Real-Time PCR Master Mixes (Thermo Fisher Scientific). Ct values of target genes were normalized using that of housekeeping geneGapdh. In addition, we employed a reference gene Rpl13a used for normalization in qRT-PCR analyses in AP mouse tissue [33]. The primer sequences used in the study are listed in Supplementary Table 1.
Flow cytometric analysis of cell proliferation and apoptosis
Cell apoptosis was detected using the Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences) following the manufacturer’s instructions. Briefly, the cells were collected and suspended in binding buffer, followed by incubation with Annexin V-FITC and PI. The stained samples were loaded on FACSCalibur flow cytometer (BD Biosciences) and produced data were analyzed with CELLQuest software (BD Biosciences). Cell proliferation was analyzed using Click-iT® Plus EdU Proliferation Kits (ThermoFisher) according to the manufacturer's protocol.
ChIP-qPCR
Chromatin immunoprecipitation (ChIP) was performed using the EZ ChIPTM Chromatin Immunoprecipitation Kit (Merck Millipore, Billerica, MA, USA) according to the manufacturer’s instruction. Cells were cross-linked with 1% formaldehyde for 15 min and quenched using 125 mM glycine for 5 min. Sonication was performed by three runs of 10 cycles (30 s “ON”, 30 s “OFF”) at high power setting (Bioruptor, Diagenode). Chromatin lysates were immunoprecipitated with anti-H3K27ac (ab4729, Abcam). Real-time quantitative PCR was using PCR master mix (Bio-Rad) and specific primers for regulatory elements regions of human IL-6 listed in Supplementary Table 1.
Western Blot Assay
The primary antibodies used for Western blotting are as following: ARID1A (1:1000, Cell Signaling, #12354), IL-6 (1:1000, Abcam, ab6672), and β-actin (1:5000, Santa Cruz, AC-15). After incubation with peroxidase-conjugated secondary antibodies, membranes were developed using the enhanced chemiluminescent immunoassay for the detection of antigen.
Statistical Analysis.
All data from mice experiments are shown as means ± SEM. Statistical comparisons were performed by the two-tailed Student’s t-test. Analyses were performed using GraphPad Software (San Diego, CA, USA). p < 0.05 was considered statistically significantly.