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. Author manuscript; available in PMC: 2024 Oct 1.
Published in final edited form as: Cancer Res. 2024 Apr 1;84(7):1084–1100. doi: 10.1158/0008-5472.CAN-23-2659

Fig. 6. MCL-1 plays an important role in ONC213-induced cell death.

Fig. 6.

a-c. MV4–11 cells were treated with vehicle or ONC213 for 48 h (panel a) or up to 24 h (panels b&c). Whole cell lysates were analyzed by western blot and probed with the indicated antibodies. The fold changes for the densitometry measurements, normalized to β-actin and then compared to vehicle control, are shown below the corresponding blots. Relative MCL-1 protein levels are graphed in panel c. d. MV4–11 cells were treated with vehicle or ONC213 for up to 24 h and then analyzed by Annexin V-FITC/PI staining and flow cytometry analysis. ** P<0.01; *** P<0.001 compared to vehicle control at the same timepoint. e. MV4–11 cells were treated with vehicle, ONC213, MG132, or ONC213 + MG132 for 8 h. Whole cell lysates were analyzed by western blot. The fold changes for the densitometry measurements, normalized to β-actin and then compared to vehicle control, are indicated. f. MV4–11 cells were treated with vehicle or ONC213 for 8 h. Total RNA was extracted, and real-time RT-PCR was performed. The relative changes in MCL-1 transcripts, normalized to GAPDH, in comparison to control samples were quantified. The displayed results represent the mean of three independent experiments, with fold changes calculated via the comparative Ct method. g-i. MV4–11 cells were treated with vehicle or ONC213 for 8 h, washed and then treated with 10 μg/mL cycloheximide for up to 2 h. Western blots were generated utilizing whole cell lysates and representative blots are shown in panel g. The fold changes for the densitometry measurements, normalized to β-actin and then compared to 0 min, are graphed in panel h. MCL-1 protein half-life was calculated using GraphPad Prism 9.0 and graphed in panel i. j. MV4–11 cells were treated with vehicle, ISRIB, ONC213, or ISRIB combined with ONC213 for 8 h. Whole cell lysates were analyzed by western blot. The fold changes for the densitometry measurements, normalized to β-actin and then compared to vehicle control, are indicated. k. MV4–11 cells were treated with vehicle, ONC213, ISRIB, or ONC213 combined with ISRIB for 24 h and then analyzed by Annexin V-FITC/PI staining and flow cytometry analysis. *** P<0.001 compared to ONC213 treatment alone.