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X. Li et al.
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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 indi- cated 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 extra- long isoforms, respectively. The fold changes for the Mcl-1 or Bim densitometry measurements, normalized to β-actin and then compared to no drug treatment con- trol, 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.
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haematologica | 2021; 106(5)