Fig 2: WFDC3 enhances the inhibitory effects of estrogen on migration and EMT.a and b Transwell migration and Matrigel invasion assays performed in LoVo cells transfected with WFDC3 plasmid (a) or SW480 cells transfected with pooled WFDC3 siRNA (b) and treated with estrogen or DMSO control. The bar graph indicates the number of migrated cells. c–e Western blot analysis of ZO-1, N-cadherin, E-cadherin, Vimentin, and Snail protein levels in RKO cells (c), LoVo cells (d) transfected with WFDC3 plasmid, and in SW480 cells treated with pooled WFDC3 siRNA (e). f and g The epithelial marker E-cadherin and mesenchymal marker N-cadherin were detected by immunofluorescence staining in LoVo cells (f) and SW480 (g) cells treated as in a and b. Nuclei were counterstained with Hoechest 33342 (blue). Data are expressed as mean ± SD of at least three independent experiments. Statistics were performed using unpaired Student’s t-test, ***P < 0.001.

Fig 3: WFDC3 expression in CRC tissues.a Representative images of WFDC3 immunohistochemistry (IHC) staining in CRC tissues and adjacent normal tissues. b Representative images of negative and positive expression of WFDC3 in CRC tissues (Magnification, 200×, Scale bars, 100 µm). c The number of WFDC3-positive and WFDC3-negative expression in adjacent normal tissues and CRC tissues. Statistical significance was determined by Pearson ?2-test. d Quantification of WFDC3 expression according to IHC scores in normal and tumor tissues. IHC scores were compared by unpaired Student’s t-test. e WFDC3 protein expression in four paired CRC tissues and adjacent normal tissues was assessed by western blot. Western blot image has been cropped for presentation. f and g Relative mRNA expression of WFDC3 (f) and ERß (g) in representative paired CRC tissues and adjacent normal tissues (blue: normal; red: tumor). Statistics were performed using paired Student’s t-test. h Kaplan–Meier analysis of overall survival in CRC patients categorized according to WFDC3 status. i Kaplan–Meier analysis of disease-free survival in CRC patients categorized according to WFDC3 status.

Fig 4: The effects of WFDC3 in estrogen-induced inhibition of metastasis are dependent on ERß levels.a and b Early estrogen response (a) and late estrogen response (b) signaling was enriched in the WFDC3-high group in the hallmark gene sets database according to gene set enrichment analysis (GSEA). c Western blot analysis of WFDC3 and ERß expression in CRC cell lines. d and e WFDC3 overexpression (d) or depletion (e) exerts no significant effect on ERß mRNA expression levels. f WFDC3 overexpression increased the ERß protein level in a plasmid concentration-dependent manner in RKO and LoVo cells. g WFDC3 depletion downregulates the protein levels of ERß in HCT116 and SW480 cells. h and i Transwell migration and Matrigel invasion assays of the WFDC3-overexpressing LoVo cells treated with PHTPP (h) or a siERß pool (i) in the presence of estrogen. j and k Rescue effects of treatment with estrogen combined with PHTPP (j) or a siERß pool co-transfection (k) on WFDC3 overexpression-mediated inhibition of cell migration in LoVo cells determined by wound healing assays. The bar graph indicates the number of migrated cells and percentage of wound closures. Data are expressed as mean ± SD of at least three independent experiments. Statistics were performed using unpaired Student’s t-test, ***P < 0.001.

Fig 5: ERß and WFDC3 attenuates CRC cell invasion through the suppression of TGFBR1.a ERß overexpression inhibits mRNA expression of TGFBR1, SMAD2, and SMAD3. b TGFBR1 overexpression rescues the ERß-mediated reduction in SMAD2/3 protein expression levels. c Matrigel invasion assays of CRC cells co-transfected with Flag-TGFBR1 and Myc-ERß or control plasmid. d Effect of SB525334 on ERß depletion-induced SMAD2/3 downregulation in LoVo cells. e Effect of galunisertib on ERß depletion-induced cell migration in LoVo cells. f WFDC3 overexpression inhibits mRNA expression of TGFBR1, SMAD2, and SMAD3. g TGFBR1 overexpression rescued the WFDC3-mediated decrease in SMAD2/3 protein expression levels. h Matrigel invasion assays of CRC cells co-transfected with Flag-TGFBR1 and Flag-WFDC3 plasmids. The bar graph indicates the number of invaded cells. i Effect of SB525334 on WFDC3 depletion-induced SMAD2/3 downregulation in LoVo cells. j Effect of galunisertib on WFDC3 knockdown-induced cell migration in LoVo cells. Bar graphs indicate the relative protein levels compared with ß-actin or the number of migrated cells. Data are expressed as mean ± SD of at least three independent experiments. Statistics were performed using unpaired Student’s t-test, **P < 0.01, ***P < 0.001.