Piericidin

Intact cell oxygen consumption and extracellular acidification rates (OCR and ECAR) were determined using a Seahorse XFe96 analyser (Agilent). A375 cells were seeded at 1.5 × 10⁴ cells in 100 ml per well in 96-well Seahorse cell culture plates, in DMEM supplemented with 10% FBS (Gibco no. 26140-079) and penicillin/streptomycin (Gibco no. 10378-016). After 14 h, 80 ml of media was removed and 160 ml of HEPES-buffered Seahorse DMEM (Agilent no. 103575-100) supplemented with 10 mM glucose (Sigma no. G8270), 1 mM pyruvate (Agilent 103578-100) and 2 mM glutamine (Agilent 103579-100) was added, and the plate was transferred to a 37 °C non-CO₂ incubator for 1 h. The Seahorse cartridge was hydrated according to the manufacturer’s protocol. oligomycin A (Sigma), BAM15 (Sigma) and piericidin A (Enzo Life Science) + antimycin A (Sigma) were prepared in Seahorse DMEM and added to the cells at final concentrations of 2 mmol l⁻¹ (oligomycin), 2 mmol l⁻¹ (BAM15) and 1 mmol l⁻¹ + 1 mmol l⁻¹ (piericidin + antimycin), respectively. Three baseline respiratory rate measurements were taken, followed by sequential injections of each inhibitor with three measurements each.