Fig. 3: GPX4 transcription is regulated by estrogen/ERβ in osteoblasts.

a Relative mRNA expression of Gpx4 in MC3T3-E1 cells treated with estrogen (17β-estradiol, 1 μM) for different time periods (n = 3 independent cell culture preparations). b Protein level of GPX4 in MC3T3-E1 cells exposed to estrogen (17β-estradiol) at doses ranging from 10 nM to 1 μM for 24 hours (n = 3 independent cell culture preparations). c Effect of estrogen antagonist fulvestrant (1 μM) on GPX4 expression treated with estrogen (17β-estradiol, 1 μM), n = 3 independent cell culture preparations. d, e Validation of Esr2 (left) and Esr1 (right) siRNA sequences by western blot. f, g Relative mRNA expression of Gpx4 in MC3T3-E1 cells after knockdown of ERβ (left) or ERα (right), respectively (n = 3 independent cell culture preparations). h Protein expression of GPX4 in MC3T3-E1 cells treated with protein synthesis inhibitor (CHX, 100 μM) in the presence or absence of estrogen (17β-estradiol, 1 μM) (n = 3 independent cell culture preparations). Data are presented as means ± SEM. i Verification of ESR2-Flag plasmid by western blot. j Effect of ERβ on the activity of GPX4-luc reporter (n = 3 independent cell culture preparations). k Transcription factor ERβ binding sequence predicted in the JASPAR database. l Schematic diagram of GPX4 luciferase promoter. Luc, luciferase. m EMSA assays showing a direct interaction between ERβ protein and GPX4 E-box element. The experiment was repeated three times independently with similar results. n Workflow of ChIP experiment. o ChIP assays showing significant enrichment of ERβ at the E-box of GPX4 (n = 3 independent cell culture preparations). p Effect of ERβ on the activity of mutant GPX4-luc reporter (n = 8 independent cell culture preparations). q Schematic diagram illustrating the estrogen/ERβ-dependent transactivation of GPX4 expression. Data are presented as means ± SD unless specific description. Statistical analysis was performed with unpaired two-tailed t-test (a, f–g, j, o, p) or one-way ANOVA with Tukey’s post hoc test (b, c), or two-way ANOVA (h). Source data are provided as a Source Data file.