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. 2017 Dec 14;118(3):388–397. doi: 10.1038/bjc.2017.432

Figure 2.

Figure 2

FP downregulates MCL-1 expression and upregulates BIM that contributes functionally to potentiation of ABT-199 lethality. (A) U266 cells were treated with ABT-199±FP for 6 h, after which immunoblotting analysis was performed to monitor the levels of MCL-1 and BCL-2 (upper panel). The ratio of BCL-2/MCL-1 was quantified by densitometry (lower panel). The results are representative of three separate experiments; (B) U266 cells were infected with shMCL-1 lentivirus particles to target MCL-1 (shMCL-1#1 using one viral dose, shMCL-1#2 using two viral doses) or control particles (shNC) according to the manufacturer’s instructions. Following 48 h infection, MCL-1 protein levels were assessed by immunoblotting (upper panel), and cells were further treated with ABT-199 (500 and 750 nM) for further 24 h. Cell death was analysed by flow cytometry after staining with 7-AAD, with knockdown cells showing MCL-1 downregulation and significantly greater death than control cells (lower panel). The results are representative of three separate experiments; (C) U266 cells were infected with lentivirus encoding Cas9 and sgRNA targeting GFP or CDK9. Following 48 h infection, cells were treated with ABT-199 (500 and 750 nM) for 24 h. Immunoblotting analysis was carried out to monitor p-CTD(S2), p-CTD(S5), CDK9, MCL-1, BCL-2, and cleaved PARP; (D) U266 cells were incubated with varying concentrations of ABT-199±FP (150 nM) for 12 h. Immunoblot analysis was performed to monitor p-CTD(S2), p-CTD(S5), RNA Pol II, MCL-1, BCL-2, Bik, and cleaved PARP (left panel). Meanwhile, NOXA, PUMA, BMF, HRK, BCL-XL, and three isoforms (EL, L, and S) of BIM were monitored (right panel); (E) U266 cells were stably transfected with constructs encoding shRNA targeting BIM (shBIM) or scrambled sequence as a negative control (shNC). Cells were treated with ABT-199 (750 nM)±FP (150 nM) for 12 h. Immunoblot analysis was carried out to monitor the three isoforms (EL, L, and S) of BIM, p-CTD(S2), p-CTD(S5), MCL-1, BCL-2, and cleaved caspase-3 and PARP. α-tubulin or β-actin were assayed to ensure equivalent loading and transfer.