Fig. 2. Metabolic reprogramming contributes to GPX4 regulation.

A H446, H1688, A549, and H1299 were treated with Etoposide (25 µM) for the indicated time, Western blot was used to determine HIF-1α, GPX4 protein levels. B Co-transfection of the HRE promoter reporter gene with control Renilla luciferase reporter gene was done in indicated cells. 24 h later cells were treated with Etoposide (30 µM) for 24 h, and luciferase activity was measured and normalized using dual luciferase reporter system and the bars represent the mean ± S.D. of triplicates (*p < 0.05, **p < 0.01, for difference from untreated control by ANOVA with Dunnett’s correction for multiple comparisons, ns means no statistical difference). C Western blot examines MCT4, Glut1, HK2 expression in cells treated with different doses of Etoposide. D Indicated cells were untreated, or treated with Etoposide (30 µM) for 48 h and siHIF-1 α for 24 h then treated with Etoposide (30 µM) . Western blot was used to determine HIF-1 α, MCT4, Glut1, HK2 protein levels. E Cells were treated with Etoposide and then extracellular lactate concentration was measured by Lactate Colorimetric/Fluorometric Assay Kit (Biovision). (**p < 0.01, ***p < 0.001 for Difference from control cells by ANOVA with Dunnett’s correction for multiple comparisons, ns means no statistical difference). F Measurement of 2-DG uptake in cells after exposed etoposide for 5 h by Glucose Uptake Kit (*p < 0.05, **p < 0.01, for difference from untreated control by ANOVA for multiple comparison, ns means no statistical difference). G Western blot examines GPX4 expression in cells treated with different concentrations of lactate. H Western blot examines GPX4 expression in cells treated with different concentrations of glucose. I 48 h after transfection with control siRNA or LDHA siRNA, cells were treated with different concentrations of glucose. Western blot examines GPX4 expression. J Western blot analysis of GPX4 in cells treated with glucose in the presence of 2-DG.