Drug Sensitivity and Apoptosis Assays

Cells were seeded at 1,000 cells per well in 96-well plates in triplicates and treated after 24 hours. After 5–7 days of treatment (only 1 dose exposed to cells after the cells are plated for 24 h), cells were washed twice with PBS and lysed with deionized-water for 1 hour at 37 °C. Cells were stained with PicoGreen (Invitrogen), a fluorescent dye that selectively binds double stranded DNA, for 2 hours in dark at RT, as previously described¹⁸. The intensity of the fluorescent signal correlates to the number of viable or surviving cells. Results were analyzed using GraphPad Prism (GraphPad Software Inc.), La Jolla, CA.

For apoptoic assay, cells were seeded at 10⁶ cell density in 10 cm dishes and treated with MMC (150 nM/L) and/or MK-1775 (400 nM/L) for 24 hours. Annexin V13242 labeling kit (Invitrogen) was used following manufacturer’s protocol to measure the apoptotic cells. Samples were analyzed on flow cytometry BD LSRII (BD Biosciences, San Jose, CA) and 50,000 events were recorded for each sample in Fig. 2A and 10,000 events were recorded for each sample in Fig. 2C. Each cell line has its own compensation controls and gating was done according to each cell line unstained/untreated sample. Gating strategy is shown in Supplementary Fig. S2C. Only V+/PI− cells are considered as apoptotic cells and plotted in the graph. Data was analyzed in FlowJo (FlowJo LLC.).