Fig. 2
ERRα inhibition induces AML cell death through intrinsic apoptosis. A Venn diagram showing the overlap between down-regulated genes by XCT-790 treatment in RNA-Seq and ERRα + genes among HALLMARK OXPHOS genes. Four genes selected for further experimental validations were marked in red. B Relative expression of NDUFS3, UQCRFS1, COX5A, and COX5B significantly downregulated by XCT-790 treatment (10 µM for 24 h) in AML patient-derived cells (n = 9). C. Western analysis of mtOXPHOS complexes in THP-1 cells by XCT-790 treatment (5 µM; for lanes 2 and 3, 24 and 48 h, respectively) and at multiple concentrations (2.5 µM, 5 µM, 10 µM; 48 h). D Oxygen consumption rate (OCR) evaluated by Seahorse XF analysis between wild-type and ERRα knockout (KO #13 and #20) cells. E Representative electron microscopic images between wild-type (WT) and ERRα KO KG1α cells. Damaged, swollen, and disturbed cristae in the mitochondria of ERRα KO KG1α cells are marked with arrows. Scale bars, 1 µM and 0.2 µM. Quantification of the cristae width between WT (n = 20) and ERRα KO (n = 18) cells (right). F, G, and I CCK8 assay for KG1α cells (F, I), patient-derived AML cells, and primary monocytes from healthy controls (HC) (G). F and G, XCT-790 for 72 h; I, XCT-790 and/or Z-VAD-FMK (Z-VAD) for 30 h. H Western analysis of apoptotic proteins in KG1α cells by XCT-790 treatment (5 µM; for lanes 2 and 3, 24 and 72 h, respectively) and at multiple concentrations (2.5 µM, 5 µM, 10 µM; 72 h). J Progression of tumor volumes in NOD/SCID mice subcutaneously injected with KG1α cells transduced with targeting ERRα (shERRα-KG1α) or non-targeting control shRNA lentivirus (shNS-KG1α). K. Survival rates of NIG mice injected with shERRα-KG1α or shNS-KG1α (4 × 106 cells/mice). Median survival times are 56 and 36 days for the shERRα-KG1α-engrafted mice (n = 10) and the shNS-KG1α-engrafted mice (n = 14), respectively. L Flow cytometric analysis of engrafted HL-60 cells into NOD/SCID mice at 4 weeks post-transplantation. A representative image of the engrafted HL-60 cells (human CD45+ (hCD45+) and murine CD45− (mCD45−)) by XCT-790 (8 mg/kg) for three weeks (left); the quantitative data of tumor burdens in the bone marrows (right). P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***) were used to determine statistically significant differences. Two-tailed t test (B, E right, L right), extra sum of square F test (G, J), log-rank test (K) or one-way ANOVA (F, I). Data are the combined results from three independent experiments (K), representative of three independent experiments (C, E left, H, and L left). Data represent means ± SD from three or four independent experiments performed in triplicate (B, D, E right, F, G, I, J, and L right)