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. 2020 Jul 9;10(18):8098–8110. doi: 10.7150/thno.45363

Figure 1.

Figure 1

Mcl-1 inhibitors restores apoptosis response and sensitivity to regorafenib in Mcl-1-KI HCT116 cells. (A) MTS analysis of wild-type (WT) and Mcl-1-KI HCT116 cells treated with regorafenib at indicated concentrations with or without a combination with an indicated Mcl-1 inhibitor (5 μM) for 72 hours. (B) Crystal Violet staining of WT and Mcl-1-KI HCT116 cells treated with regorafenib (40 μM), an indicated Mcl-1 inhibitor (5 μM), or their combination for 48 hours. (C) Colony formation of WT and Mcl-1-KI HCT116 cells treated with regorafenib (40 μM), S63845 (5 μM), or their combination for 48 hours. Left panel: representative pictures of colonies visualized by crystal violet staining 2 weeks after treatment; right panel: enumeration of colony numbers. (D) Combination index (CI) and fraction affected of regorafenib and S63845 combining at different concentrations in WT and Mcl-1-KI HCT116 cells treated for 48 hours (shown in Figure S1B-C) were analyzed by the CompuSyn program (ComboSyn). (E) Apoptosis in cells treated as in B was analyzed by counting condensed and fragment nuclei after nuclear staining with Hoechst 33258. (F) Western blotting of Mcl-1 and cleaved (C) caspases 3, 8 and 9 in cells treated as in C. (G) Cellular thermal shift assay (CETSA) for the binding of indicated inhibitors to endogenous Mcl-1 in HCT116 cells. Cells were treated with the control DMSO or an indicated Mcl-1 inhibitor (5 μM) for 2 hours, followed by heating of cell lysates at indicated temperatures for 3 minutes and probing of supernatants by Mcl-1 western blotting. (H) Immunoprecipitation (IP) analysis of the binding between endogenous Mcl-1 and PUMA in cells treated as in C for 24 hours. In A, C and E, results were expressed as means ± s.d. of three independent experiments. **, P <0.01; ***, P <0.001.