ERα is endocytosed through a caveolin 1–dependent pathway. (a) Top: Confocal images of MCF7 cells treated for 15 min as indicated and stained for caveolin 1. Bottom: Merge between caveolin 1 signal and differential interference contrast (DIC) images. Arrows indicate internal or peripheral localization of caveolin 1. (b) Top: Confocal images of MCF7 cells, treated for 15 min as indicated, and stained for clathrin. Bottom: Merge between caveolin 1 signal and DIC images. Arrows indicate internal or peripheral localization of clathrin. (c) Confocal images of MCF7 cells treated for 15 min as indicated and stained for caveolin 1 or EEA1. Arrows indicate regions of colocalization between the two markers. (d) Western blots of MCF7 cells transfected with siRNAs against caveolin 1, clathrin HC, or scrambled for 48 h. Blotting antibodies are shown on the right. Fold change relative to scrambled siRNA is shown on the bottom. (e) Luciferase assay in MCF7 cells transiently transfected with pTK-ERE-Luc and pTK-Renilla and the respective siRNAs and treated for 14 h as indicated. Differences between groups were analyzed by two-way ANOVA followed by Bonferroni contrasts adjusted for multiple comparisons (n = 3 replicates). Data are represented as mean ± SD. **, P < 0.01. Shown data are representative of at least three independent experiments performed. Treatments: ethanol (vehicle) or 10−8 M E2, 2.5 µg/ml filipin, 5 µM PAO. Bars, 10 µm.
