Cmrl 1066 medium

Regularly cycling females underwent ovarian hyperstimulation beginning on days 1–4 of menses. Monkeys were administered 30 IU recombinant human follicle stimulating hormone (rhFSH) (IVF Prescription, Puregon) intramuscularly, twice-daily at twelve hour intervals for 11–11.5 days. An injection of 1000 IU recombinant human chorionic gonadotropin (hCG; IVF Prescription, Ovidrel) was administered in the evening on day 11 or 11.5. Laparoscopic oocyte aspirations were performed between 38–40 h post hCG injection in which the ovaries were manipulated to visualize and aspirate all visible follicles. Oocytes were aspirated into HTF-HEPES solution (Irvine Sci., cat no: 90126) supplemented with 3 mg/mL human albumin (MP Biomedicals, cat no: 823051), 0.28 mg/mL heparin (Sigma-Aldrich, cat no: H3149) and 0.28 mg/mL hyaluronidase (Sigma-Aldrich, cat no: H3884) and filtered through a 100 μM strainer (PluriSelect, cat no: 435010051) to remove blood clots and cumulus cells from oocytes. The oocytes were washed from the strainer and placed into maturation medium composed of CMRL 1066 medium (Thermo Fisher Scientific, cat no: 11530037) supplemented with 0.5 mM sodium pyruvate (Sigma-Aldrich, cat no: 2256), 2 mM Alanyl-glutamine (Sigma-Aldrich, cat no: G8541) and 20% fetal bovine serum (FBS; Peak Serum, cat no: PS-FB1, Wellington, CO, USA) as similarly described by Curnow and Hayes^{21 (link)}.

The mouse insulin-secreting cell line MIN6 was cultured exactly as previously described [32 (link)]. The siRNA duplexes directed against HDAC1 (si-HDAC1), HDAC3 (si-HDAC3), and GFP (si-GFP) were introduced using the Lipofectamine 2000 (Invitrogen AG) exactly as described [33 (link)]. Human pancreases were harvested from adult brain-dead donors in accordance with French regulations and with the local Institutional Ethical Committee from the “Centre Hospitalier Régional et Universitaire de Lille.” Pancreatic islets were isolated after ductal distension of the pancreas and digestion of the tissue as described previously [34 (link)]. All experiments were carried out at least on islets cells of >80% purity. Purified islets were cultured in CMRL 1066 medium (Gibco BRL, Life Technologies) containing 0.625% free fatty acid HSA (Roche Diagnostics), penicillin (100 μUI/mL), and streptomycin (100 μg/mL). A pool of 4 siRNAs was used to knock down HDAC1 and HDAC3 expression (ON-TARGETplus SMARTpool, Thermo Scientific Dharmacon).

The rat insulin-secreting cell line INS-1E was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) (PAA laboratories, GE Healthcare, Velizy-Villacoublay, France), 1 mM sodium pyruvate, 50 μM β-mercaptoethanol, and 10 mM Hepes [31 (link)]. Human pancreases were harvested from adult brain-dead donors in accordance with French regulations and with the local Institutional Ethical Committee from the “Centre Hospitalier Régional et Universitaire de Lille.” Pancreatic islets were isolated after ductal distension of the pancreas and digestion of the tissue as described previously [32 (link)]. All experiments were carried out at least on islets with a purity of and viability >80%. Purified islets were cultured in CMRL 1066 medium (Gibco BRL, Life Technologies) containing 0.625% free fatty acid human serum albumin (Roche Diagnostics), penicillin (100 μUI/mL), and streptomycin (100 μg/mL). The siRNA duplexes directed against JNK3 (siJNK3) or siRNA control against GFP (siGFP) were previously described [29 –31 (link)]. The siRNA duplexes were introduced using the Lipofectamine 2000 (Life Technology, Saint Aubin, France) as described [29 , 30 (link)].

Isolated islets (from the same groups as above) recovered overnight in CMRL-1066 medium (Gibco) containing 10% fetal bovine serum and 2 mmol/l L-glutamine at 37 °C. Thirty islets per NHP were placed in a dynamic perifusion system (Amersham Biosciences AKTA FPLC System) as previously described^{37 (link)}. To summarize, the perifusion was performed using Krebs buffer with 2.8 mM glucose at a flow rate of 1 ml/min for 30 min to establish stable basal insulin secretion. Next, the islets were perifused with 2.8 mM glucose for 10 min, and fractions of 500 μl were collected every 30 s. Then, the glucose concentration was increased to 20 mM, and fractions of 500 μl were collected every 30 s for 20 min. Finally, the islets were perifused with 30 mM KCl, and fractions of 500 μl were collected every 30 s for 10 min. After the perifusion, the islets were recollected from the column for protein quantification. The insulin in the effluent was measured as described above. The fractional insulin secretion rate was calculated as secreted insulin per minute normalized to the protein content.

Human islets were obtained from adult, nondiabetic organ donors from Prodo Laboratories, Inc., or the Integrated Islet Distribution Program at City of Hope. Islets were cultured in CMRL 1066 medium (Gibco) supplemented with 10% FBS (MilliporeSigma), 10 mM HEPES (AmericanBio), 2 mM l-glutamine (MilliporeSigma), and 1% pen-strep (Gibco). Where indicated, islets were treated with 25 or 100 ng/mL IFN-γ (R&D Systems) or 10 ng/mL TNF-α (R&D Systems). In some experiments, islets were pretreated with 5 μM ruxolitinib (Selleckchem) for 1 hour prior to the addition of IFN-γ. Islets were harvested at indicated time points and dissociated into single-cell suspensions using 0.05% trypsin-EDTA (Gibco). Cells were stained with FluoZin-3 (Invitrogen) and TMRE (Life Technologies) for β cell isolation experiments and sorted using a FACSAria II (BD).