Fig. 3

GSTZ1 suppresses the Warburg effect and ROS accumulation in HCC cell lines. a-b The ECAR and OCR in GSTZ1-overexpressing Huh7 cells (left) and GSTZ1-KO HepG2 cells (right) were determined using the Seahorse XF Cell Energy Phenotype test. GSTZ1-overexpressing Huh7 cells were less glycolytic than AdGFP control cells. In contrast, GSTZ1-KO HepG2 cells were more glycolytic than parental cells. a OCR versus ECAR. b ECAR values. c Representative fluorescence images (left) and quantification (right) of ROS levels in GSTZ1-overexpressing SK-Hep1 (top) and GSTZ1-KO HepG2 cells (bottom) stained with the CellROX Orange fluorescence probe. Immunofluorescence intensity was quantified using ImageJ. Values represent mean ± SD values. d Western blotting revealed the levels of 4-HNE modification in proteins in GSTZ1-overexpressing Huh7 cells (top) and GSTZ1-KO HepG2 cells (bottom). e GSSG/GSH ratio for GSTZ1-overexpressing Huh7 cells (top) and GSTZ1-KO HepG2 cells (bottom) determined with Grx1-roGFP2. f Liver GSH levels in wildtype and Gstz1^−/− mice, detected via mass spectrometry. For Western blotting, 50 μg protein was loaded per well. Values represent mean ± SD values (n = 3, performed in triplicate), *p < 0.05, **p < 0.01, Student’s t-test (two groups) or one-way ANOVA followed by Tukey tests (three groups). ECAR, extracellular acidification rate; OCR, oxygen consumption rate; HCC, hepatocellular carcinoma; ROS; reactive oxygen species; GSH, glutathione; GSSG: GSH disulfide