Pre warmed accutase

Cells were seeded in 12-well plates in duplicate or triplicate (60 000 cells/1 ml media/well) and treatments were administered the day after. Cells were treated with perhexiline (7 μM), gemcitabine (1 μM) or the combination for 24 h. For lipid peroxidation detection, cells treated with erastin (2 μM) were used as positive controls. After treatments, the media was supplemented with either the BODIPY 581/591 C11 probe (lipid peroxidation sensor), CellROX Orange (total ROS) or MitoSOX (superoxide anion) at a final concentration of 2, 5 and 10 μM, respectively. Cells were incubated for 30 min (BODIPY and CellROX) or 20 min (MitoSOX) at 37°C, then harvested with pre-warmed accutase (Gibco) and resuspended in PBS for flow cytometry analysis. Five thousand events per sample were acquired in a MACSQuant-VYB (Miltenyi Biotec), and data analysis was done with the FlowJo software.

Panc-1 cells (400,000) were plated in 6-well plates in DMEM with 10% FBS. The day after, four CPT1C siRNA (L-008824-01-0010, ON-TARGETplus siRNA Reagents, Dharmacon) were transfected at a final concentration of 20 nM using INTERFERin reagent (Polyplus-transfection) with Opti-MEM (Gibco) medium according to the manufacturer’s protocol. A control siRNA pool (Scramble siRNA) was used as the negative control (D-001810-10-05, ON-TARGETplus Non-targeting Pool, Dharmacon). The medium was replaced with DMEM 10% FBS 6h after the start of transfection. To check the downregulation of CPT1C expression after 24h, cells were detached with pre-warmed accutase (Gibco), triplicates were pooled and processed for RT-qPCR as described above. For the viability test, cells were treated the day after transfection with 7μM of perhexiline in triplicates. Cells were detached 72h later and viability was assessed via Trypan blue exclusion using a cell viability analyzer (Vi-cell XR, Beckman Coulter).