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. 2023 Aug 10;12:e80494. doi: 10.7554/eLife.80494

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).

Figure 5—source data 1. Raw data for Figure 5.

Figure 5.

Figure 5—figure supplement 1. No significant oxidative-stress was raised by application of E2 and iron within the indicated concentration.

Figure 5—figure supplement 1.

(A) SOD2 protein levels of J774a.1 post treatments with FAC/DFP in the presence/absence of E2. (B) The enzymatic activity of catalase in J774a.1. n = 4. (C) Relative ERα mRNA expression of J774a.1 treated with different concentrations of FAC in the presence/absence of E2, assessed by qPCR. n = 5. Student’s t-test analysis was used for (B, C).
Figure 5—figure supplement 1—source data 1. Raw data for Figure 5—figure supplement 1.
Figure 5—figure supplement 2. E3-ligase responses to iron and E2 treatment in different cell types.

Figure 5—figure supplement 2.

(A) BRCA1 and AHR protein expressions in J774a.1 were detected. (B) The protein expression of ERα and its related E3 ligase, BRCA1 and AHR, was detected in MCF-7 cell line. n = 4.
Figure 5—figure supplement 2—source data 1. RAW data for Figure 5—figure supplement 2.