The ERRγ inverse agonist DN200434 increases mitoROS levels, and decreases mTORC1 activity and GPX4 protein levels. (A, B) Representative fluorescence images showing mitochondrial superoxide production in sorafenib-resistant (SR) Huh7 (A) and SK-Hep1 (B) cells. The bar graph shows quantification of MitoSOX fluorescence intensity (red) and nuclear counterstaining with DAPI (blue). (C) Measurement of relative GSH/GSSG ratios in sorafenib-resistant (SR) Huh7 and SK-Hep1 cells. (D) Levels of phosphorylated mTOR, phosphorylated 4E-BP1, and GPX4 in sorafenib-resistant (SR) Huh7 and SK-Hep1 cells treated with sorafenib or DN200434 in the presence/absence of GSH. Scale bar, 20 μm. Data are expressed as the mean ± SEM of three independent experiments. N.S., not significant; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Sora, Sorafenib; DN, DN200434.