Fig. 2.

E2 regulates CLDN6 expression via ERβ. a Western blot analysis of ERα and ERβ expression in MCF-7 cells treated with E2. Actin served as a loading control. b Western blot analysis of ERβ in MDA-MB-231 cells treated with E2. c MDA-MB-231 cells were transfected with either negative control (sh-NC) or three different ERβ shRNAs for 48 h and were then subjected to western blot analysis to detect the protein abundance of ERβ. Actin was used as the loading control. d ERβ knockdown abolished the CLDN6 expression induced by E2. e MDA-MB-231 cells were incubated with DPN for 24 h at the indicated concentration. CLDN6 gene and protein expression levels were detected by using semiquantitative RT-PCR and western blot. f Immunofluorescence of CLDN6 (red) was prominent along the edges of the MDA-MB-231 cells upon DPN treatment. Nuclei were stained with 4, 6-diamino-2-phenylindole (DAPI) (blue) (scale bar, 20 μm). g Tight junctions (white arrowheads) between cells were prominent in MDA-MB-231 cells after DPN treatment as observed by TEM. h The migration (scale bar, 200 μm) and invasion (scale bar, 50 μm) abilities of MDA-MB-231 cells treated with DPN were decreased. i CLDN6 knockdown rescued the migration and invasion abilities of MDA-MB-231 cells after DPN treatment. The ERβ antagonist PHTPP (10 μM, 24 h) (j) and ERβ knockdown (k) abolished the DPN-induced CLDN6 expression. Overexpression of ERβ induced CLDN6 upregulation in SR-BR-3 (l) and MDA-MB-231 (m) cells after treatment with DPN. Data are presented as mean ± SD. The data shown are representative results of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001