Figure 5. The interactive effects of iron overload and E2 treatment on estrogen receptor α (ERα) downregulation are mediated by the E3 ligase MDM2.
(A) Evaluation of ERα proteasome-dependent degradation in J774a.1 cells by western blotting. MG132: 10 μM. n = 4. (B) ERα turnover rate in J774a.1 cells under FAC or E2 + FAC conditions, detected by western blotting after 20 μM cycloheximide (CHX) treatment. *p<0.05 using two-way ANOVA. (C) Ubiquitination of ERα, evaluated by western blotting (anti-ubiquitin) following immunoprecipitation against ERα antibody. n = 3, *p<0.05. (D) Relative Mdm2 mRNA expression in J774a.1 cells, assessed by qPCR, n = 4, **p<0.01. (E) The protein levels of ERα in the presence of FAC or FAC plus E2 in J774a.1 cells after treatment of Nutlin-3, a specific antagonist of Mdm2. n = 3. (F) The protein levels of ERα in the presence of FAC or FAC plus E2 in human umbilical vein endothelial cells (HUVECs) after treatment of Nutlin-3. n = 3. (G) Mdm2 protein expression in the aortas of mice in the early postmenopausal (EPM) or late postmenopausal (LPM) stage, as detected by western blotting. n = 3/group. (H) MDM2 protein levels in patient plaques, detected by western blotting and quantified with ImageJ. n = 4/group, ***p<0.001. Two-way ANOVA was used for (B). Student’s t-test analysis was used for (C, D H).