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. 2020 Mar 12;106(5):1262–1277. doi: 10.3324/haematol.2019.233445

Figure 3.

Figure 3.

CUDC-907 upregulates Bim and downregulates Mcl-1 enhancing venetoclax activity against acute myeloid leukemia cells. (A) Acute myeloid leukemia (AML) cells were treated with vehicle control, venetoclax (VEN), CUDC-907 (CUDC), or in combination for up to 24 hours. Flow cytometry analysis of Annexin-V-FITC/PI staining was performed. Results are shown as mean percent Annexin V+ cells ± standard error of the mean (SEM). ***P<0.001 compared to single drug treatments. (B, C) AML cells were treated with vehicle control, venetoclax, CUDC-907, or in combination for 24 hours, and whole cell lysates were subjected to western blotting. Representative western blots are shown. The fold changes for the densitometry measurements, normalized to b-actin and then compared to no drug control, are indicated below the corresponding blot. Bim S, L, and EL indicate Bim short, long, and extra-long isoforms, respectively. (D, E) U937 and MOLM-13 cells were treated for 24 hours with vehicle control, venetoclax, CUDC-907, or in combination. Bcl-2 (left panel) or Bim (right panel) was immunoprecipitated from whole cell lysates. Western blots were probed with anti-Bim, -Bcl-2, or -Mcl-1 antibody. The fold changes for the densitometry measurements, normalized to b-actin and then compared to no drug control, are indicated below the corresponding blot. *Indicates the light chain of Bim or Bcl-2 antibody. (F) U937 cells were infected with NTC- (U937/NTC) or Bim-shRNA (U937/Bim) (panel F) or Precision LentiORF Mcl-1 (U937/Mcl-1) or RFP control (U937/RFP) (panel G) lentivirus particles overnight, then washed and incubated for 48 hours prior to adding puromycin or blasticidin, respectively, to the culture medium. The antibiotic-resistant cells were treated with vehicle control, venetoclax, CUDC-907, or in combination for 24 hours. Whole cell lysates were subjected to western blotting. Bim S, L, and EL indicate Bim short, long, and extralong isoforms, respectively. The fold changes for the Mcl-1 or Bim densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated (top panel). Annexin V/PI staining and flow cytometry analysis results are shown (bottom panel). ***P<0.001 compared to NTC or RFP.