Bay 61–3606 downregulated Mcl-1 expression by inhibition of CDK9 and RNA polymerase II phosphorylation.

(A) Cells were treated by Bay 61–3606 (2.5 μM) combined with TRAIL (50 ng/ml) for 6 h, and transcriptional downregulation of Mcl-1 was measured by RT-PCR (A, upper), and Mcl-1 luciferase reporter assay (A, lower). (B) MCF-7 cells were transfected with scrambled siRNA or three different CDK9 siRNAs (siCDK9 #1, #2, and #3). After 48 h of transfection, the expressions of CDK9, RNA pol II, Mcl-1 with phosphorylation of RNA pol II were analyzed by Western blotting. (C) Cell extracts were prepared from cells treated with in (A) and the expressions of CDK9, RNA pol II, Mcl-1 with phosphorylation of RNA pol II were analyzed by Western blotting. (D) MCF-7 cells were transfected with pcDNA3-WT-CDK9-HA or pcDNA3-DN-CDK9-HA. After 48 h of transfection, cells were treated with Bay 61–3606 (2.5 μM) for 6 h. Cell extracts were then analyzed by Western blotting using the indicated antibodies. Values are the mean ± SE from three separate experiments performed in triplicate. Asterisks indicate significant differences compared with the control (** P-values < 0.01, and *** P-values < 0.001).