Fig. 4.

ERα expression is higher in LAM tissue fibroblasts of both WT and Arom^hum, which contributes to the higher estrogen-responsive gene expression in LAM tissue of Arom^hum mice. (A) Relative mRNA levels of ERα in UAM, LAM, and QM of WT and Arom^hum mice. Two-way ANOVA with Tukey’s multiple comparison test. *P < 0.05. n = 10 mice in each group. (B) ERα mRNA levels and (C) ERα protein levels in primary fibroblasts from UAM and LAM of WT mice. The data shown in C are representative of three independent experiments. Two-tailed Student’s t test, **P < 0.01, n = 3. (D) ERα localization in UAM and LAM of WT and Arom^hum mice was measured by IHC staining. (Scale bar, 50 μm.) (E) A minimum of 1,000 nuclei were counted in sections to calculate the average H-score of ERα in fibroblasts of UAM and LAM from WT and Arom^hum mice. Two-way ANOVA with Sidek’s multiple comparison test, *P < 0.05, **P < 0.01, n = 10. mRNA levels of Pgr, Greb1, Ccnd1, and Adora1 in tissue lysates (F, G, H, and I; n = 9) and primary fibroblasts (J, K, L, and M; n = 6) of UAM and LAM from WT and Arom^hum mice. mRNA levels of Pgr and Greb1 in LAM primary fibroblasts from Arom^hum mice after MPP (10 µM) treatment (N and O) or siRNA-mediated knockdown of ERα (P and Q) in the presence or absence of E₂ (10 nM). Cells were pretreated with MPP for 2 h before the addition of E₂. Veh, vehicle. Two-way ANOVA with Sidak’s multiple comparison test, *P < 0.05, **P < 0.01. GAPDH mRNA or protein levels served as controls.