Human samples

Rapid harvesting of cadaveric pancreatic tissues was obtained with informed consent from next of kin, from heart-beating, brain-dead donors, with research approval from the Human Research Ethics Committee at St Vincents Hospital, Melbourne. Pancreas from individuals without and with diabetes, islet, acinar and ductal samples were obtained as part of the research consented tissues through the National Islet Transplantation Programme (at Westmead Hospital, Sydney and the St Vincents Institute, Melbourne, Australia), HREC Protocol number: 011/04. The donor characteristics of islet cell donor isolations are presented in Table 1.

Islets were purified by intraductal perfusion and digestion of the pancreases with collagenase AF-1.24 (SERVA/Nordmark, Germany) followed by purification using Ficoll density gradients.25 Purified islets, from low-density gradient fractions and acinar/ductal tissue, from high-density fractions, were cultured in Miami Media 1A (Mediatech/Corning 98021, USA) supplemented with 2.5% human serum albumin (Australian Red Cross, Melbourne, VIC, Australia), in a 37C, 5% CO2 incubator.

Total RNA from human ex vivo pancreatic cells was isolated using TRIzol (Invitrogen) and RNeasy Kit (QIAGEN) including a DNase treatment. First-strand cDNA synthesis was performed using a high-capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturers instructions. cDNA primers were designed using oligoperfect designer (Thermo Fisher Scientific), as shown in Table 2. Briefly, quantitative RT-PCR analyses were undertaken using the PrecisionFast 2 qPCR Master Mix (Primerdesign) and primers using Applied Biosystems 7500 Fast Real-Time PCR System. Each qPCR reaction contained: 6.5l qPCR Master Mix, 0.5l of forward and reverse primers, 3.5l H2O and 2l of previously synthesised cDNA, diluted 1/20. Expression levels of specific genes were tested and normalised to 18s ribosomal RNA housekeeping gene.

Modification of Histone H3 and histone-associated Ezh2 protein signals were quantified in human pancreatic ductal epithelial cells (AddexBio) by the LI-COR Odyssey assay. The cells were treated with 5 or 10M of GSK 126 (S7061, Selleckchem) for 48h. Histones and their associated proteins were examined using an acid extraction and immunoblotting as described previously.18 Protein concentrations were determined using Coomassie Reagent (Sigma) with BSA as a standard. Equal amounts (3g) of acid extract were separated by Nu-PAGE (Invitrogen), transferred to a PVDF membrane (Immobilon-FL; Millipore) and then probed with antibodies against H3K27me3 (07449, Millipore), H3K27ac (ab4729, Abcam), H3K9me3 (ab8898, Abcam), H3K9me2 (ab1220, Abcam), H3K4me3 (39159, Active Motif), Ezh2 (#4905, Cell Signaling Technology), and total histone H3 (#14269, Cell Signaling Technology). Protein blotting signals were quantified by an infra-red imaging system (Odyssey; LI-COR). Modification of Histone H3 and histone-associated Ezh2 signals were quantified using total histone H3 signal as a loading control.

Chromatin immunoprecipitation assays in human exocrine cells were performed previously described.26,27 Cells were fixed for 10min with 1% formaldehyde and quenched for 10min with glycine (0.125M) solution. Fixed cells were resuspended in sodium dodecyl (lauryl) sulfate (SDS) lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.1) including a protease inhibitor cocktail (Roche Diagnostics GmBH, Mannheim, Germany) and homogenised followed by incubation on ice for 5min. Soluble samples were sonicated to 200600bp and chromatin was resuspended in ChIP Dilution Buffer (0.01% SDS, 1.1% Triton X-100, 1.2mM EDTA, 16.7mM Tris-HCl pH 8.0, and 167mM NaCl) and 20l of Dynabeads Protein A (Invitrogen, Carlsbad, CA, USA) was added and pre-cleared. H3K27me3 antibody was used for immunoprecipitation of chromatin and incubated overnight at 4C as previously described.28 Immunoprecipitated DNA were collected by magnetic isolation, washed low salt followed by high salt buffers and eluted with 0.1M NaHCO3 with 1% SDS. Protein-DNA cross-links were reversed by adding Proteinase K (Sigma, St. Louis, MO, USA) and incubation at 62C for 2h. DNA was recovered using a Qiagen MinElute column (Qiagen Inc., Valencia, CA, USA). H3K27me3 content at the promoters of the INS, INS-IGF2, NGN3 and PDX1 genes were assessed by qPCR using primers designed from the integrative ENCODE resource.29 ChIP primers are shown in Table 3.

Insulin and glucagon localisation in human islets were assessed using paraffin sections (5m thickness) of human pancreas tissue fixed in 10% neutral-buffered formalin and stained with hematoxylin and eosin (H&E) or prepared for immunohistochemistry. Insulin and glucagon were detected using Guinea Pig anti-insulin (1/100, DAKO) or mouse anti-glucagon (1/50) mAbs (polyclonal Abs, Sigma-Aldrich).

Pharmacological inhibition of EZH2, human pancreatic exocrine cells were kept untreated or stimulated with 10M GSK-126 (S7061, Selleckchem) at a cell density of 1105 per well for 24h. After 24h of treatment, fresh Miami Media was added to the cells, which were treated again with 10 GSK-126 and cultured for a further 24h. All cell incubations were performed in Miami Media 1A (Mediatech/Corning 98-021, USA) supplemented with 2.5% human serum albumin (Australian Red Cross, Melbourne, VIC, Australia), in a cell culture incubator at 37C in an atmosphere of 5% CO2 for 48h using non-treated six-well culture plates (Corning).

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Inhibition of pancreatic EZH2 restores progenitor insulin in T1D donor | Signal Transduction and Targeted Therapy - Nature.com