Cell Viability Assay for MCL-1 Inhibitors

Human cell lines were originally sourced from the American Type Culture Collection (ATCC) and were authenticated by Promega GenePrint 10 System (Promega WI, USA). Cells were maintained at 37 °C with 5% CO2 with 10% fetal bovine serum (FBS), except for in vitro experiments using A1210477 where FBS level was reduced to 3% during drug treatment and also in the relevant control samples. Cell viability was determined by CellTiter 96 MTS assay (Promega) after 48 h incubation with the indicated concentration of MCL-1 inhibitor UMI-77 (Selleck, UK), S63845 (Apexbio, UK) or A1210477 (Apexbio, UK). SYTOX Green (Invitrogen, UK) was used to identify dead cells and cell confluence measured using the Incucyte Live Cell Analysis System (Essen Bioscience, UK). 10 μM etoposide (Sigma, UK) was used to induce apoptosis and 10 μM Q-VD-OPh (Apexbio, UK) was used to block caspase activity. CRISPR/Cas9 gene editing using the LentiCRISPRv2 system (Addgene, MA, USA) was performed for BAX and BAK as described previously^{2 (link)}.