Fig 2: FOXC1 enhances c-MYC expression to promote gastric cancer cell proliferation through activation of the Wnt signaling pathway. (A) TopFlash/FopFlash assays using normal gastric cells (GES-1) and gastric cancer cells (AGS, SGC-7901, MGC-803, MKN-45, HGC-27). (B) qPCR results in FOXC1 KO and FOXC1-overexpressing AGS and MKN-45 cells indicated that mRNA levels of ß-catenin remained unchanged. (C) The results of western blot assays in FOXC1 KO and FOXC1-overexpressing AGS and MKN-45 cells indicated that protein levels of ß-catenin remained unchanged (ß-catenin Antibody: CST #8480S). (D) FOXC1 KO increased ß-catenin levels in the cytoplasm but decreased them in the nucleus (ß-catenin Antibody: CST #8480S). (E) Immunofluorescence assay in FOXC1 KO and FOXC1-overexpressing AGS and MKN-45 cells showed that the level of ß-catenin translocation into nucleus changed with knockout and overexpression FOXC1 (ß-catenin Antibody: CST #8480S). (F) FOXC1 overexpression inhibit the phospho-ß-Catenin (Ser33/37/Thr41) in AGS and MKN-45 cells (phospho-ß-Catenin (Ser33/37/Thr41) Antibody: CST #9561T). (G,H) co-IP assays and immunofluorescence assay indicated that FOXC1 binds to the ß-catenin protein in the cytoplasm (ß-catenin Antibody: CST #8084S; FOXC1 Antibody: Abcam #ab227977). (I) Western blot assays and immunoprecipitation assay were applied to confirmed the distribution of FOXC1 in the nucleus. (J) co-IP assay was applied to confirmed the distribution of phospho-ß-Catenin (Ser33/37/Thr41) in the nucleus (phospho-ß-Catenin (Ser33/37/Thr41) Antibody: CST #9561T). (K) co-IP assay was applied to confirmed whether FOXC1 bound to the ß-catenin protein in the cytoplasm and nucleus. Immunoprecipitation assay results showed that FOXC1 bound to non-phospho-ß-Catenin in the cytoplasm (ß-catenin Antibody: CST #8084S; phospho-ß-Catenin (Ser33/37/Thr41) Antibody: CST #9561T; Non-phospho-ß-Catenin (Ser33/37/Thr41) Antibody: CST #8814T), and FOXC1 did not bound to ß-Catenin in the nucleus (ß-catenin Antibody: CST #8084S). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: WSB1 promotes ß-catenin nucleus translocation and inhibits its ubiquitination. (A) Bel-7402 cells were co-transfected with vector or pCDH-WSB1 and pGL4.14-TCF/LEF1-luciferase for 48 h. Luciferase activity was normalized by cell numbers and shown as fold change. *P < 0.05 (versus vector group). (B) H460 cells were transduced with lentivirus WSB1 shRNA followed by transfection with TCF/LEF1-luciferase for 48 h. Luciferase activity was normalized by cell numbers and shown as fold change. **P < 0.01, ***P < 0.001 (versus control group). (C) Bel-7402 cells were infected with lentivirus pCDH Vector or lentivirus pCDH-WSB1 for 72 h and collected for cell fraction (nuclei and cytoplasm) separation. Cellular localization of ß-catenin was detected by Western blotting in cell fraction. GAPDH was used as loading control for cytoplasm fraction and lamin B was for nucleus fraction. (D) Immunofluorescence staining of ß-catenin in Bel-7402 cells infected with lentivirus pCDH vector or lentivirus pCDH-WSB1. (E) Bel-7402 cells infected with lentivirus pCDH vector or lentivirus pCDH-WSB1 were treated with WNT3a condition medium (WNT3a CM) for 16 h, and stained with ß-catenin antibody by immunofluorescence. (F) H460 cells infected with lentivirus vector or lentivirus WSB1 shRNAs were treated with WNT3a condition medium (WNT3a CM) for 16 h, and stained with ß-catenin antibody by immunofluorescence. (G) Relative mRNA level of WSB1 and CTNNB1 in Bel-7402 cells infected with lentivirus pCDH-WSB1 or 72 h. (H) Relative mRNA level of WSB1 and CTNNB1 in H460 cells infected with lentivirus WSB1 shRNA for 72 h. (I) 293FT cells were infected with lentivirus pCDH vector or lentivirus pCDH-WSB1 or lentivirus pCDH-?SOCS for 72 h. Then the cells were plated and transfected with pCDNA-3.0-ß-catenin-HA and His-Ub for 48 h and treated with MG132 (10 µmol/L) for 8 h before collecting cells. Then the cells were lysed and for immunoprecipitated with anti-HA antibody followed by immunoblotting with anti-ubiquitination and anti-HA antibody. Data are represented as mean ± SD, n = 3; statistical significance was determined by Student's t-test. n.s., no significant difference (P > 0.05).

Fig 4: Overexpressing Sp5 partially mimics the effect of CHIR in medium with 2i. (A) Flow chart showing the method used to identify candidate genes downstream of CHIR and Stat3. (B) qRT-PCR analysis of Socs3 and Sp5 expression induced by CHIR, PD03 and 2i (left), or LIF (right) for 12 h. Data represent mean±s.d. of three biological replicates. **P<0.01 versus NT. NT, no treatment. (C) FLAG-tagged Sp5 was introduced into 46C mESCs and the protein level of FLAG-tagged SP5 was determined by western blotting. a-tubulin is used as a loading control. (D,E) Alkaline phosphatase staining images of PB-Sp5 mESCs cultured in N2B27 medium with PD03 for the indicated number of passages (P). Scale bars: 100 µm. (F–H) qRT-PCR analysis of the indicated gene expression after treatment with CHIR for the indicated time in 46C mESCs, CTNNB1-KO (ß-catenin knockout) and Tcf3-KO mESCs. CHX, cycloheximide treatment. Data represent mean±s.d. of three biological replicates. **P<0.01 versus 0 h CHIR.

Fig 5: Requirement of ß-catenin signaling for sustaining HCC cell growth. (A) HCC cell lines were transiently transfected with CTNNB1 siRNAs for 72 hours. Cell lysates were collected for Western blotting with indicated antibodies. Tubulin and ß-actin served as loading control (LC). (B) Silencing of ß-catenin caused the reduction of ß-catenin signaling activity (mean ± SD, n = 2, two times). (C) Silencing of ß-catenin inhibited the growth of HCC cell lines as determined by MTT assay (mean ± SD, n = 4, two times). (D) Silencing of ß-catenin alters cell cycle progression in Huh6 (all phases significantly changed) and Huh7 cells (G2/M phase significantly reduced). SNU449 is less clearly altered, although the proportion of cells in S-phase is reduced (mean ± SD, n = 2, two times). (E) ß-catenin knockdown reduced cell growth in colorectal cancer cell lines. Values depicted are relative to the ones obtained with the nontargeting siRNA that are arbitrarily set to 1. *P < .05; **P < .01.