Hy b0764

In vitro, each group repeated independently for 3 times (n = 3). And all experimental groups received 6 h of oxygen-glucose deprivation and 18 h of reoxygenation (29 (link)). CGS-21680 (A2aR specific agonist, 30μM, Tocris Bioscience, 1036) and dbcAMP (selective PKA activator, 5μM, MedChemExpress, HY-B0764) were added 1 h before reoxygenation. H89 (the PKA selective inhibitor, 10 μM, MedChemExpress, HY-15979) was used 5 min before CGS21680. To verify the effect of autophagy on cell survival, autophagy agonist Rapamycin (100 nM, MedChemExpress, HY-10219) and antagonist 3-Methyladenine (3-MA, 10 mM, MedChemExpress, HY-19312) were used 1 h before reoxygenation (38 (link), 39 (link)).
In vivo study, 36 adult male rats were randomized into six groups (n = 6): Sham group, IR group (30 min LAD occlusion and 120 min reperfusion, 1% DMSO in 1 ml saline, iv), IR+CGS21680 group (30 μg/kg 5min before reperfusion and 30 μg/(kg·min) for 1 h, i.v.), and IR+ZM241385 group (A2aR antagonist, 0.2 mg/kg 5 min before reperfusion, i.v.), IR+dbcAMP group (5 mg/kg, 5 min before reperfusion, i.v.) (40 (link)), and IR+CGS21680+H89 group (20 mg/kg, 5 min before CGS21680, i.v.) (41 (link)).

Human trophoblast cell lines (Jeg3, HTR8) and human decidual stromal cell line (hESC) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Jeg3 and HTR8 cells were cultured in a 5% humidified carbon dioxide atmosphere at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (Gibco, Life Technologies, Grand Island, NY, USA) with 10% foetal bovine serum (Gibco, Life Technologies, Grand Island, NY, USA), 50 mg/mL streptomycin, and 50 U/mL penicillin. Jeg3 is the only trophoblast cell line that expresses human leukocyte antigen G (HLA-G), which is gradually expressed during development from CTBs to EVTs.
hESCs were cultured in a 5% humidified carbon dioxide atmosphere at 37 °C in phenol red-free Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (Meilunbio, Dalian, China) with 10% foetal bovine serum, 50 mg/mL streptomycin, and 50 U/mL penicillin. One micromolar medroxyprogesterone-17-acetate (MPA) (HY-B0469, MedChemExpress, NJ, USA) and 0.5 mM N6,20-O-dibutyryladenosine cAMP sodium salt (db-cAMP) (HY-B0764, MedChemExpress, Monmouth Junction, NJ, USA) were added to the culture for 6 days to induce hESC decidualization in vitro [14 (link)].

MIN6 cells (4*10⁴ cells per well) and isolated islets (10 islets per well) were cultured in 48-well plate and changed the culture medium every 24 h. Following preincubation for 1 h in glucose-free Krebs–Ringer bicarbonate HEPES buffer (KRBH, 119 mM NaCl, 4.74 mM KCl, 2.54 mM CaCl 2,1.19 mM MgCl 2,1.19 mM KH 2 PO 4, 25 mM NaHCO 3, 10 mM HEPES, pH 7.4), the MIN6 cells or islets were treated for 1 h in KRBH buffer with low glucose (2 mmol/L for MIN6 cells, 3.3 mmol/L for islets) or high glucose (20 mmol/L for MIN6 cells, 16.7 mmol/L for islets) or high KCL (50 mM) with or without lorcaserin, SB242084(2901, Tocris Bioscience), Bay K8644 (B112, Sigma-Aldrich) or db-cAMP (HY-b0764, MedChem Express LLC). For experiments involving pertussis toxin (PTX, P7208, Sigma-Aldrich) treatment, MIN6 cells were pretreated with PTX (150 ng/ml) overnight in the culture medium as described previously (Parandeh et al., 2020 (link)). The supernatants were then obtained for determination of insulin concentration. Intracellular insulin contents were extracted in acid–ethanol solution [74% (vol./vol.) ethanol, 1.4% (vol./vol.) HCl] overnight at 4°C. The insulin levels were measured by radioimmunoassay (RIA) kit (North Biological Technology Research Institute of Beijing) as described previously.