Fig. 4

TET1-mediated demethylation of RSPO3 inhibits CCA growth. A–C Overexpression of TET1, TET2 and TET3 in QBC939 cells confirmed by RT-qPCR. Data are shown after normalization to β-actin. D Expression of RSPO3 in TET1, TET2 and TET3 overexpression QBC939 cells. Data are shown after normalization to β-actin. E Knockdown of TET1 in TFK1 cells resulted in lowered expression of RSPO3 confirmed by RT-qPCR. Data are shown after normalization to β-actin. F An illustration of targeted methylation by sgRNA-dCas9TET1CD. G Differential DNA methylation targeting RSPO3 promoter methylation in CCA cell lines QBC939 and HCCC9810. H, I RSPO3 expression after targeted RSPO3 promoter methylation in CCA cell lines QBC939 and HCCC9810. J, K Cell proliferation after methylation of the targeted RSPO3 promoter in the CCA cell lines QBC939 and HCCC9810 was performed using Cell Counting Kit-8 (CCK-8) assay. L–N Cell proliferation after methylation of the targeted RSPO3 promoter in the CCA cell lines QBC939 and HCCC9810 was performed using clone formation assay