Fig. 5. ONC213 induces a mitochondrial stress gene expression signature and suppresses protein synthesis.

a&b. MV4–11 cells were treated with vehicle or ONC213 for 4 or 8 h. 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 indicated below the corresponding blots (upper panels). Densitometry results from 3 independent experiments are graphed and shown in the lower panels. ns, not significant; * P<0.05; ** P<0.01; *** P<0.001 compared to vehicle control. c-g. MV4–11, MOLM-13, U937 and THP-1 cells were treated with vehicle or 500 nM ONC213 for 48 h and then RNAseq gene expression profiling was performed. The top 50 differentially expressed upregulated genes are shown in panel c as a heat map. Activated transcription factors were determined by unbiased interrogation by the interactive pathway analysis tool, Enrichr, using the 100 upregulated differentially expressed genes (panel d). GSEA of the 100 upregulated genes by ONC213 compared to genes associated with Clpp knockout is shown in panel e. GSEA of ONC213 upregulated genes overlapping with HtrA2 knockout is shown in panel f. GSEA of ONC213 upregulated genes overlapping with mitochondrial disorders is shown in panel g. Enrichr analysis of the ONC213 signature genes affecting translation is shown in panel h. i. MV4–11 cells were treated with vehicle or ONC213 for up to 16 h. Whole cell lysates were analyzed by western blot. j. MV4–11 cells were treated with vehicle, cycloheximide (CHX; as a positive control for inhibition of protein synthesis), ONC213, ZVAD-FMK, or ONC213 + ZVAD-FMK for 8 h and then 1 μM puromycin was added for 30 min. Whole cell lysates were analyzed by western blot and probed with anti-puromycin antibody. k. MV4–11 cells were treated with vehicle, ISRIB, ONC213, or in combination for 8 h. 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 indicated below the corresponding blots. l. MV4–11 cells were treated with vehicle, cycloheximide, ONC213, ISRIB, or ONC213 combined with ISRIB for 8 h and then 1 μM puromycin was added for 30 min. Whole cell lysates were analyzed by western blot and probed with anti-puromycin antibody. One representative blot is shown (left panel). Densitometry measurements (normalized to vehicle control) are graphed and shown (right panel). *** P<0.001.