Triacsin c

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.