2.5. Cell Proliferation/Viability Assays and Washout Experiments

Proliferation/viability of cultured cells was measured by CellTiter96 Non-radioactive cell proliferation assay (Promega; Madison, WI). Briefly, various wild type and mutant BRAF melanoma cells were seeded in 96 well plates and treated with pevonedistat, vemurafenib or the combination pevonedistat and vemurafenib at various concentrations. Control cells were treated with DMSO. 96 h following treatment, cells were stained with the dye solution according to the manufacturer's protocol. Absorbance was recorded at 570 nm and growth curves were established. To test the effect of transient exposure of melanoma cells to pevonedistat on rereplication and growth inhibition, we conducted the washout experiments where melanoma cells or PIG3V melanocytes were treated with 1 μM pevonedistat for different times (4, 8, 12 and 24 h) before the drug was washed out by washing the cells 2 × with PBS, and adding drug-free fresh growth media to cells. Cells were counted every 24 h by Countess Automated Cell Counter (Invitrogen), and harvested at the indicated times for PI staining and FACS analysis (cell cycle profile) or for immunoblotting.