Evaluating Cell Viability under Stress

Cell viability was measured by CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to manufacturer’s instructions after exposing 3 days under different conditions including low-glucose (0.45 g/l glucose)^{10 (link)}, hypoxia (1% oxygen)^{7 (link)}, BMI1 inhibitor (PTC596, PTC therapeutics; PTC209, PTC therapeutics), and EZH2 inhibitor (EPZ6438, Medchemexpress LLC; DZNep, SELLECKCHEM). 1×10⁶ cells were seeded for cell viability under low-glucose or/and hypoxia conditions, 2×10³ cells were seeded for drug response under BMI1 or EZH2 inhibitor treatment. IC50 values of BMI1 or EZH2 inhibitors were calculated with GraphPad Prism software. For competitive response assay of BMI1 and EZH2 inhibitors, the proportion of GFP or mCherry positive cells was determined by flow cytometry after treatment the co-cultured MES83-GFP and PN528-mCherry cells with PTC-209, and DZNep. Two-dimensional titration assay was performed on four different subtype GSCs after combined treatment with different doses of PTC596 and EPZ6438. Relative cell growth was measured over a four-day time course using CellTiter-Glo Luminescent Cell Viability Assay kit (Promega) after seeding 1×10³ cells.