Supplementary Methods and Supplementary Figure 1-20 from SIX1 Promotes Tumor Lymphangiogenesis by Coordinating TGFβ Signals That Increase Expression of VEGF-C

Supplementary Methods and Supplementary Figure 1-20: Supplementary Figure 1 shows the growth of the tumors after orthotopic transplantation. Supplementary Figure 2 shows that over-expression of SIX1 promotes lymphangiogenesis and lymph-node metastasis of C33a cells in vivo. Supplementary Figure 3 shows the assay of cytokine and growth factor with antibody-based array. Supplementary Figure 4 shows the expression of the genes coding for the lymphangiogenic factors. Supplementary Figure 5 shows that SIX1 promotes VEGF-C expression in C33a cells. Supplementary Figure 6 shows that SIX1 promotes migration and tube-formation of HLECs. Supplementary Figure 7 shows the knock-down of VEGF-C in cervical cancer cells. Supplementary Figure 8 shows that the promotional effect of SIX1-expressing tumor cells on the migration and tube-formation of HLECs in vitro is mediated by VEGF-C. Supplementary Figure 9 shows that the promotional effect of SIX1-expressing tumor cells on lymphangiogenesis in vivo is mediated by VEGF-C. Supplementary Figure 10 shows that the promotional effect of SIX1-expressing C33a cells on lymphatic-vessel is mediated by VEGF-C. Supplementary Figure 11 shows that the promotional effect of SIX1 on lymph-node metastasis in vivo is mediated by VEGF-C. Supplementary Figure 12 shows the differences in gene expression levels between tumors in vivo and tumor cells cultured in vitro. Supplementary Figure 13 shows the immunohistochemical analysis of TGF-βs in tissue microarray of human CSCC specimens. Supplementary Figure 14 shows that SIX1 enhances the effect of TGF-β1 on VEGF-C production by tumor cells and tube-formation of HLECs in vitro. Supplementary Figure 15 shows that TGF-β contributes to the differences in VEGF-C expression between tumors in vivo and tumor cells cultured in vitro. Supplementary Figure 16 shows that TGF-β signaling is required for SIX1 to promote lymphangiogenesis and lymph-node metastasis in vivo. Supplementary Figure 17 shows that knocking down the expression of SMAD2 or SMAD3 or TβR1 suppresses TGF-β1 induced VEGF-C expression. Supplementary Figure 18 shows that higher expression of SIX1 correlates with higher expression of TβR1 in human CSCC specimens. Supplementary Figure 19 shows the binding of SIX1 to the VEGF-C promoter. Supplementary Figure 20 shows that knocking down the expression of SMAD2 and SMAD3 does not affect the binding of SIX1 to the VEGF-C promoter.