Fig. 9

Mcl-1 and c-Myc are key mediators of ouabain-indued apoptosis in AML cells. A, B AML HL-60 cells were treated with ouabain (0 − 100 nM) for 48 h. A Western blot analysis of pro- and cleaved (active) caspase-3 (C3), caspase-8 (C8), and caspase-9 (C9) levels. Quantitative analysis of caspase activation as a ratio of cleaved to pro-C8 and C9 levels by densitometry is shown. Data are mean ± SD (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus nontreated control cells; two-sided Student's t test. B Western blot analysis of the key apoptosis regulatory proteins, including Mcl-1, c-Myc, c-FLIP_L, and c-FLIP_S, and their densitometric analysis. β-actin was used as a loading control. Data are mean ± SD (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus nontreated control cells; two-sided Student's t test. C HL-60 cells were genetically knocked down with sgRNA sequence against MCL1 in the DOX-inducible CRISPR/Cas9 system. (upper) Western blot analysis showing Mcl-1 knockdown efficiency in iMcl-1/Cas9 cells after DOX treatment (1 μg/mL). (lower) Percentage of apoptosis as evaluated by Annexin V/7-AAD assay following ouabain treatment (0 − 100 nM) with or without DOX for 24 − 48 h. Data are mean (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus nontreated control cells; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus ouabain-treated iMcl-1/Cas9 cells without DOX; two-sided Student’s t test. D Depletion of c-Myc was performed in HL-60 cells using shRNA sequence against MYC. (upper) Western blot analysis showing c-Myc knockdown efficiency in shMYC cells. (lower) Percentage of apoptosis as evaluated by Annexin V/7-AAD assay following ouabain treatment (0 − 100 nM) for 24 − 48 h. Data are mean (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus nontreated control cells; ^##P < 0.01, ^###P < 0.001 versus ouabain-treated shCON cells; two-sided Student’s t test