Fig. 6: WFDC3 promotes ERβ-mediated repression of TGFBR1 transcription.

a ERβ depletion rescues the WFDC3-mediated reduction of TGFBR1 and SMAD2/3 at the protein levels. Bar graphs indicate the relative protein levels compared with β-actin. b Western blot analysis of ERβ in nucleus and cytoplasmic fractions of RKO and LoVo cells transfected with WFDC3 or control. Histone H3 and β-actin were used as nucleus and cytoplasmic markers, respectively. c Two potential ERβ-binding sites were predicted on the TGFBR1 promoter region. ChIP assay was performed in LoVo. d ChIP assay-coupled to qPCR to analyze the effects of WFDC3 on the binding of ERβ to the promoter of TGFBR1. e The activity of the TGFBR1 promoter was determined by luciferase reporter assays in LoVo cells with WFDC3 overexpression and PHTPP treatment. f Luciferase reporter assay indicated the effects of ERβ and WFDC3 on TGFBR1 promoter (wild type or mutated) in LoVo cells with or without ERβ or WFDC3 overexpression. Firefly luciferase activity was measured and normalized to Renilla luciferase activity. Data are expressed as mean ± SD of at least three independent experiments. Statistics were performed using unpaired Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001. g A schematic model illustrates the potential mechanisms by which WFDC3 facilitates estrogen-induced inhibition in metastasis through the ERβ/TGFBR1 axis. Created with Figdraw.com.