Fig. 4

ONC213 induces a mitochondrial stress response and its combination with venetoclax diminishes mitochondria function in AML cells. (A) MV4-11, THP-1, and MV4-11/VEN + AZA-R cells were treated with venetoclax (VEN), ONC213 (ONC), or ONC + VEN for up to 48 h and then stained with annexin V/PI and analyzed via flow cytometry. *** indicates p < 0.001 compared to control and single drug treatments. (B) MV4-11, THP-1, MV4-11/VEN + AZA-R cells were treated with VEN and ONC, alone or in combination, for 4–8 h. α-ketoglutarate dehydrogenase (α-KGDH) activity was measured. The ratio of α-KGDH activity to total protein was determined and normalized to the vehicle control. ** p < 0.01 and *** p < 0.001 compared to control. ### p < 0.001 compared to VEN. ns not significant compared to ONC. (C) MV4-11, THP-1, and MV4-11/VEN + AZA-R cells were treated with VEN and ONC, alone or in combination, for 4–8 h. Representative western blots of whole cell lysates probed with the indicated antibodies are shown. The fold changes for the densitometry measurements, normalized to β-actin and then compared to vehicle control, are indicated below the corresponding blots. (D) MV4-11, THP-1, and MV4-11/VEN + AZA-R cells were treated with VEN and ONC, alone or in combination, for 4–8 h and then subjected to the cellular mito stress test. OCR was measured following injection with oligomycin A (complex V inhibitor), FCCP (mitochondrial membrane uncoupler), and rotenone + antimycin A (complex I and III inhibitors). (E) MV4-11, THP-1, and MV4-11/VEN + AZA-R cells were treated with VEN and ONC, alone or in combination, for 4–8 h and then stained with JC-1 and analyzed via flow cytometry for aggregate vs. monomer fluorescence ratio. *** p < 0.001 compared to control. ## p < 0.01 and ### p < 0.001 compared to ONC alone and VEN alone. (F) MV4-11/VEN + AZA-R and THP-1 cells were cultured in either glucose or galactose containing media for 48 h and then treated with VEN, ONC, or the combination of ONC + VEN for 24 h. Then the cells were stained with annexin V/PI and analyzed via flow cytometry for apoptosis. ns not significant and *** p < 0.001 compared to the same treatment in glucose media