Xestospongin c

Wide-field time-lapse imaging was performed for 4 days, the medium was switched to DMEM containing 0.1% DMSO or inhibitors [10 μM dantrolene (Supelco; Merck KGaA, Darmstadt, Germany), 100 μM ryanodine (Sigma-Aldrich), 1 μM TTX (Abcam plc., Cambridge, UK), 10 μM Xestospongin C (Abcam plc.)], and imaging was continued for another 4 days.

All chemicals and inhibitors were dissolved according to the manufacturer's instructions to indicated concentrations at stock solutions. Working concentrations are indicated for each experiment. m–3M3FBS was prepared at 10 mM stock solution and purchased from Sigma–Aldrich (catalog no.: 525185). Xestospongin C prepared at 1 mM stock solution and purchased from Abcam (catalog no.: ab120914). LysoTracker Red DND-99 purchased from Thermo Fisher Scientific at 1 mM. Gö6976 purchased from Sigma (catalog no.: 365250-500UG) and prepared at 1 mM. Calcineurin inhibitor VIII, CN585, was purchased from Calbiochem (CAS 1213234-31-1) and prepared at 1 mM.

INS-1 cells (kindly provided by Wollheim C) were cultured in RPMI-1640 containing 11.2 mM glucose and 2 mM glutamine supplemented with 10% FBS, 1 mM pyruvate, 10 mM HEPES, 50 μM 2-mercaptoethanol, 100 U/mL penicillin and 100 μg/mL streptomycin. INS-1 cells were seeded onto wells of Poly-L-ornithine-coated plates. 1.1B4 cells obtained from ECACC (Salisbury) through Abdul Majid FA were cultured in RPMI-1640-10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. For drug treatment, the following concentrations were used: 100 nM rotenone (Sigma), 200 nM oligomycin (Millipore), 125 nM antimycin A (Sigma), 400 μM PA (Sigma), 50 μM BAPTA-AM (Invitrogen), 20 μM ML-SI3, 200 μM GPN (Sigma), 3 μM Xesto-spongin C (Abcam), 10 μM Dantrolene (Sigma), 10 μM TPEN (Sigma), 10 μM BTP2 (Sigma), 2 mM EGTA (Sigma), 5 mM N-acetylcysteine (Sigma) and 100 μM MitoTEMPO (Sigma), 2 μM Thapsigargin (Sigma), 10 μM DC260126 (Cayman), 10 μM Fumonisin B1 (Sigma), 10 μM Myriocin (Sigma) and 5 μM Triacsin C (Abcam). PA stock solution (50 mM) was prepared by dissolving in 70% ethanol and heating at 55 °C^{40 (link)}. The working solution was made by diluting PA stock solution in 2% fatty acid-free BSA-RPMI 1640. To impose mitochondrial stress on primary pancreatic islets, higher doses of mitochondrial stressors (1 μM rotenone, 2 μM oligomycin, and 1.25 μM antimycin A) were employed.