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. 2021 Mar 4;12(3):234. doi: 10.1038/s41419-020-03313-z

Fig. 3. Gcm1 overexpression activates the Wnt/β-catenin pathway in normal C57BL/6 mESCs and NE-4C.

Fig. 3

a Increased expression of Axin2 in mESCs with Gcm1 overexpression. The western blots show that the Gcm1 overexpression plasmid increased Axin2 expression in normal C57BL/6 mESCs. PGMLV-Gcm1, Gcm1 plasmid. mESCs were transfected with either control siRNA or pGMLV-Gcm1, and the expression levels were determined 72 h post transfection by western blotting. b A Co-IP assay was used to identify the interaction between Gcm1 and β-catenin in cells transfected with pGMLV-Gcm1 overexpression plasmid in C57BL/6 mESCs. An anti-Gcm1 antibody was used for IP, and IB was performed using an anti-β-catenin antibody. c A Co-IP assay was used to identify the interaction between Gcm1 and TCF4 in cells transfected with pGMLV-Gcm1 overexpression plasmid in C57BL/6 mESCs. An anti-Gcm1 antibody was used for IP, and IB was performed using an anti-TCF4 antibody. d A Co-IP assay identified increase interactions between TCF4 and β-catenin in cells transfected with 0.5, 2, and 4 µg of pGMLV-Gcm1 for 72 h in C57BL/6 mESCs. e A Co-IP assay was used to identify the interaction between Gcm1 and TCF4, β-catenin in NE-4C cells transfected with the pGMLV-Gcm1 overexpression plasmid. An anti-Gcm1 antibody was used for IP, and IB was performed using an anti-TCF4 antibody and anti-β-catenin antibody. f The mRNA expression of Fzd5, Lrp, Wnt6, Ccnd1, Nfat5, Lef1, Tcf4, Tcf12, Bcl9L, and Axin2 in C57BL/6 mESCs transfected with the overexpression vector pGMLV-Gcm1. The mRNA was extracted from the mESCs after transfection with pGMLV-Gcm1 for 72 h. g Analysis of luciferase activity by TOP/FOP Flash assays in C57BL/6 mESCs transfected with pGMLV-Gcm1 overexpression plasmid relative to those transfected with scramble plasmid in normal and LiCl medium. LiCl (10 mmol) was added to the control medium before 72 h of transfection. Data a–g represent the mean ± SEM (n = 3). The p value was calculated by Student’s t-test, ns was for no significance, *P < 0.05, **P < 0.01, ***P < 0.001.