Induction of FOXC2 by various EMT-inducing signals. (A) Expression of FOXC2 mRNA in EpRas cells treated with TGF-β1 is shown for the indicated number of days. (B) Morphologies of HMLEs either untreated or treated with 5 ng/ml of TGF-β1 for 12 days and HMLEs expressing either the control vector, Twist, Snail, or Goosecoid, were revealed by phase-contrast microscopy. (Magnification: ×200.) (C) Expression of FOXC2 and N-cadherin was examined before, during, and after the TGF-β1 treatment. (D) The morphology of HMLEs expressing either the control vector, Twist, Snail, or Goosecoid was revealed by phase-contrast microscopy. (Magnification: ×200.) (E) Expression of FOXC2 mRNA in the HMLEs ectopically expressing either an empty vector or the indicated EMT-inducing genes by RT-PCR is shown. (F) Expression of FOXC2 protein in the HMLEs ectopically expressing either a control vector or the indicated EMT-inducing genes was examined by immunoblotting.

Expression of FOXC2 in human basal-like breast cancers. Shown are representative immunohistochemical images of human breast cancer tissue microarrays stained with the monoclonal anti-human FOXC2 antibody. (A) Normal human breast tissue. (B) Negative tumor staining. (C) Weak cytoplasmic staining. (D) Strong cytoplasmic staining. (E) Strong nuclear staining. (F) Strong nuclear and cytoplasmic staining. (Magnification: ×400.) (G) The percentage of human breast cancer samples with high level of FOXC2 expression in the respective tumor subtypes is shown.

Increased expression of FOXC2 in highly metastatic cancer cells. (A) Metastatic properties of mouse mammary carcinoma cells used for microarray analysis are represented graphically. (B) Expression of FOXC2 mRNA in primary tumors formed by individual cell lines was measured by microarray analysis. Each bar represents the mean ± SEM. (C) Expression of FOXC2 mRNA was measured by real-time PCR using RNAs isolated from individual tumors formed by the four cell lines. The expression level of FOXC2 in 67NR tumor was used as the baseline. (D) Expression of FOXC2 protein was measured in various metastatic and nonmetastatic cell lines, including the four mouse cell lines, by immunoblotting.

Effect of FOXC2 expression on the metastatic behavior of EpRas mouse mammary carcinoma cells. (A) Ectopic expression of FOXC2 protein in EpRas cells was analyzed by immunoblotting. (B) In vivo growth properties of EpRas cells expressing FOXC2 at the s.c. site of nude mice is shown. The tumor size was measured by their mean diameters ± SEM (n = 7). (C) The average number of visible lung metastatic nodules in mouse with EpRas cells expressing either the control vector or the FOXC2 is represented as mean ± SEM (n = 7). (D) The level of FOXC2 protein was assessed by immunoblotting in individual primary tumors formed by EpRas cells expressing either the control vector or FOXC2. The total number of visible lung nodules found in the respective mouse is indicated at the bottom of the gel.

Contribution of FOXC2 to the metastatic ability of mouse mammary carcinoma cells. (A) Expression of FOXC2 protein in cells expressing either the control shRNA or the FOXC2 shRNA14 is shown. (B) In vivo growth properties of 4T1 tumors expressing either the control shRNA or the FOXC2 shRNA14 grown in the mammary glands, harvested 28 days after injection, are shown. The tumor weight was measured and presented as mean ± SEM (n = 14). (C) Two representative images of lungs harvested from mice carrying 4T1 tumors expressing either the control shRNA (Left) or the FOXC2 shRNA14 (Right) are shown. (Magnification: ×0.8.) (D) The average number of lung nodules from the mice harboring 4T1 tumors expressing the control shRNA or the FOXC2 shRNA14 is shown. Each bar represents the mean ± SEM (n = 14). (E) Expression of FOXC2 protein in 4T1 cells isolated from lung nodules of mice harboring tumor from either 4T1 cells expressing control shRNA or FOXC2 shRNA is shown. β-Actin was used as a loading control.

Effects of FOXC2 expression in MDCK cells. (A) Overexpression of FOXC2 protein by retroviral infection in MDCK cells was examined by immunoblotting. (B) Phase-contrast images of MDCK cells expressing either the control vector (Left) or FOXC2 (Right) are shown. (Magnification: ×200.) (C) MDCK cells expressing either the control vector (Upper) or FOXC2 (Lower) immunostained with antibodies recognizing E-cadherin (Left) or β-catenin (Right) are shown. The green signal represents the staining of corresponding protein, and the blue signal represents the nuclear DNA staining by DAPI. (Magnification: ×200.) (D) Immunoblotting of epithelial markers, including E-cadherin, α-catenin, β-catenin, and γ-catenin in MDCK cells expressing either the control vector (Left) or the FOXC2 (Right) is shown. (E) MDCK cells expressing either the control vector (Upper) or FOXC2 (Lower) immunostained with antibodies recognizing fibronectin (Left) or vimentin (Right) are shown. The green signal represents the staining of the corresponding protein, and the blue signal represents the nuclear DNA staining by DAPI. (Magnification: ×200.) (F) Immmunoblotting of mesenchymal markers, including vimentin, fibronectin, α-smooth muscle actin, and N-cadherin in MDCK cells expressing either the control vector (Left) or the FOXC2 (Right) is shown. (G and H) The level of expression of E-cadherin, fibronectin, and N-cadherin mRNA in MDCK cells expressing control vector, FOXC2, or Snail was measured by RT-PCR (G) or real-time PCR (H). (I) Immunofluorescence images of MDCK cells expressing the control vector (Left) or the FOXC2 stained with antibodies against E-cadherin (Right). The green signal represents the staining corresponding to E-cadherin, and the blue signal represents the nuclear DNA staining by DAPI. (Magnification: ×1,000.) (J) Migration and invasion assay used MDCK cells expressing either the control vector or FOXC2. The migration and invasion ability is presented as total number of cells migrated to the bottom chamber. Each bar represents the mean ± SEM of samples measured in triplicate, and each experiment was repeated at least three times. (K) Expression of MMP2 and MMP9 was measured by ELISA; the data represent the level of expression in FOXC2 and the vector-infected control cells. Each bar is the average of the triplicate samples, and each experiment was repeated twice.