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. 2016 Jan 24;7(8):9069–9083. doi: 10.18632/oncotarget.6997

Figure 6. Maximal apoptosis induction in MCF-7 cells requires inhibition of the MYB - BCL-2 axis, MCL-1 and BCL-xL.

Figure 6

A. MYB down regulation affects BCL-2 expression but not that of MCL-1. The MYB shRNA cell line (MYB shRNA C5) and control shRNA cell line (Luc shRNA) along with wild-type MCF-7 cells were incubated without or with 5μg/ml Doxycycline for five days. Cells were harvested for western blot analysis of MYB, BCL-2 and MCL-1 using GAPDH as loading control. B. Both inhibition of BCL-2 activity and downregulation of MCL-1 are required for apoptosis induction in MCF-7 cells. Cells were treated in triplicate with either 1μM ABT-199, transfected with MCL-1 siRNA or both for 72h. Cells were harvested to estimate the amount of apoptotic cells by AnnexinV and PI staining followed by FACS analysis. C. The MYB shRNA C5 cell line was treated with or without 5μg/ml Doxycline for three days before cells were re-plated and transfected with MCL-1 siRNA as described for B.. The mean (n = 3) percentage of AnnexinV positive cells determined by flow cytometry is plotted against each treatment group. D. Expression of BCL-2, BCL-xL and BCL-W measured by q-PCR following treatment with 300nM AT7519 or 200nM BE-09-LN53 for 4h. E. Simultaneous inhibition of BCL-2, BCL-xL and MCL-1 induced maximal apoptosis. MCF-7 cells were treated with either ABT-199 (2μM) or ABT-737 (2μM) alone or in combination with MCL-1 siRNA transfection. Cells were harvested after 72h to determine the Annexin V positive cells. F. Model for the effects of CDK9i on proliferation and apoptosis of ER+veMYB+ve breast cancer cells. Survival and proliferation are driven by MYB targets BCL-2, CyclinB1, CyclinE1 and MYB-independent proteins BCL-xL and MCL-1. CDK9i induce apoptosis by inhibiting simultaneously the MYB-BCL-2 axis and MCL-1 and BCL-xL production.