Figure 5.

Synthetic lethality of non-genotoxic p53 activation and P-TEFb inhibition depends on intrinsic apoptosis pathway and on-going Pol II transcription. (A) Cytotoxicity of HCT116 and HCT116 BAX/BAK1 ^−/− cells treated with DMSO, NVP-2 (10 nM) and Nutlin-3a (10 μM) alone and in combination as indicated for 48 h measured using CellTox Green Cytotoxicity Assay. Results are presented as fluorescence values relative to the values of DMSO-treated cells and plotted as the mean ± s.e.m. (n = 3); ***, P < 0.001; n.s., non-significant, determined by Student’s t test. (B) Activity of caspase 9 measured using Caspase-Glo 9 Assay in whole cell extracts of HCT116 and HCT116 BAX/BAK1 ^−/− cells treated with DMSO, NVP-2 (3 nM) and Nutlin-3a (10 μM) alone and in combination as indicated for 18 h. Results are presented as luminescence values relative to the values of DMSO-treated cells and plotted as the mean ± s.e.m. (n = 4). **, P < 0.01; n.s., non-significant, determined by Student's t test. (C) HCT116 cells treated with indicated combinations of NVP-2 (10 nM) and Nutlin-3a (10 μM) were co-treated with DMSO and Triptolide as indicated for 24 h prior to preparation of whole cell extracts and detection of the indicated proteins by western blotting. (D) HCT116 cells were treated with DMSO, Nutlin-3a alone (10 μM) and in combination with NVP-2 (10 nM) and Triptolide (1 μM) as indicated for 24 h prior to quantifying mRNA levels of the indicated genes with RT-qPCR. Results normalized to the levels of GAPDH mRNA and DMSO-treated cells are presented as the mean ± s.e.m. (n = 3); *, P < 0.05, **, P < 0.01, ***, P < 0.001; n.s., non-significant, determined by Student’s t test. (E) HCT116 cells were treated with DMSO, Nutlin-3a (10 μM) and NVP-2 (0.5 μM) as indicated for 24 h prior to quantifying mRNA levels of the indicated genes with RT-qPCR. Results normalized to the levels of GAPDH mRNA and DMSO-treated cells are presented as the mean ± s.e.m. (n = 3); *, P < 0.05; **, P < 0.01, determined by Student's t test.