Fig. 5: Molecular mechanisms of sensitization to TRAIL–CDK9i combination.

a Dynamic BH3 profiling was performed on trametinib-sensitive and -resistant SKmel2 cells following treatment for 48 h with trametinib (10 nM) or for 12 h with a combination of dinaciclib (25 nM) and TRAIL (10 ng/ml). The Bim-derived BH3-only peptide was employed at different concentrations (0.1–3.0 μM) to assess mitochondrial apoptotic priming status. Results are expressed as Δ% priming (increase in priming compared to untreated cells). Data are mean ± SEM, n ≥ 3. b Trametinib-sensitive and -resistant SKmel2 cells were treated with trametinib (10 nM) or with dinaciclib (25 nM) for the indicated times. Cells were lysed and subjected to western blotting with antibodies specific for the indicated antigens. One representative of two independent experiments is shown. An asterisk indicates an unspecific band. c SKmel2 cells were pre-incubated with DMSO or dinaciclib (25 nM) for 1 h and subsequently treated with iz-huTRAIL at the indicated concentrations. Cell viability was quantified after 24 h (left panel). Data are mean ± SEM, n ≥ 3. SKmel2 cells were pretreated with DMSO or dinaciclib (25 nM) for 4 h and subsequently stimulated with iz-huTRAIL (10 ng/ml) for 6 h. Cells were lysed and subjected to western blotting. One representative of two independent experiments is shown (right panel). d HT29 cells were pre-incubated with DMSO or dinaciclib (25 nM) for 1 h and subsequently treated with iz-huTRAIL at the indicated concentrations. Cell viability was quantified after 24 h (left panel). Data are mean ± SEM, n ≥ 3. HT29 cells were pretreated with DMSO or dinaciclib (25 nM) for 3 h and subsequently stimulated with iz-huTRAIL (10 ng/ml) for the indicated times. Cells were lysed and subjected to western blotting (right panel). One representative of two independent experiments is shown. e HCT-116 wild-type (WT), HCT-116 Bax^−/−, HCT-116 Bak^−/− or HCT-116 Bax^−/−Bak^−/− cells were pre-incubated with DMSO or dinaciclib (25 nM) for 1 h and subsequently treated with iz-huTRAIL at the indicated concentrations. Cell viability was quantified after 24 h (left panels). Data are mean ± SEM, n ≥ 3. Representative western blots of knockout efficiency are shown (right panel).