Acute CDK9 inhibition depletes labile antiapoptotic proteins Bfl-1 and Mcl-1. (A) Western blot analysis of CDK9, CDK1, and CDK4/6 putative biomarkers and Bcl-2 family proteins in OCILY10 cells treated with the indicated doses of AZD4573 for 6 hours. Loading control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (B) OCILY10 cells were treated with 0.1 µM AZD4573, 0.1 µM dinaciclib, or 1 µM AZ’5576 for 6 and pSer2-RNAP2 and antiapoptotic biomarker expression was assessed by western blot. Loading control, GAPDH. (C) Measurement of Bfl-1 protein levels using MSD immunodetection following 6 hours of AZD4573 exposure in OCILY10 cells. Expression is normalized to DMSO-only treated cells (100%). (D) Western blot analysis of antiapoptotic Bcl-2 family protein levels in OCILY10 cells following exposure to 10 µg/mL cycloheximide (CHX) or CHX plus 10 µM MG132 for the indicated durations. MG132 treatment is indicated by “+”. Loading control, GAPDH. (E) Densiometric quantification of panel D, expression is relative to untreated (time 0 hours, 100%) cells. (F) Measurement of pSer2-RNAP2, BCL2A1 mRNA and Bfl-1 protein levels in OCILY10 cells at the indicated time points following 0.1 µM AZD4573 treatment. (G) Eµ-Myc lymphoma cells ectopically expressing BCL2A1, MCL1, BCL2, BCL2L1, or BCL2L2 prosurvival genes were treated with the CDK9 inhibitor AZ’5576 at the indicated concentrations for 24 hours. Apoptosis was measured by flow cytometric annexin V staining.