Fig 1: Characterization of SIRT2 wild-type and knockout iPSCs in vitro.Immunofluorescence of the pluripotency marker OCT4 in a WT-iPSCs and b KO-iPSCs. Scale bar = 50 µm. Immunofluorescence of the three germ layer differentiation markers in differentiated c WT-iPSCs and d KO-iPSCs assessed by embryoid body formation. Tuj1 for ectoderm, a-SMA for mesoderm, and SOX17 for endoderm. Scale bar = 100 µm. Representative images are from experiments performed in triplicate
Fig 2: Upregulation of cadherin 1 (CDH1) and SRY-box containing gene 17 (SOX17) expression in EC9706 cells by 5-azacytidine treatment. The expression of CDH1 and SOX17 was upregulated following treatment with 5-azacytidine in EC9706 cells. (A) RT-qPCR analysis of CDH1 and SOX17 expression. Compared with the vehicle control (DMSO-treated cells), the expression levels of CDH1 and SOX17 were increased approximately 7.33- and 9.30-fold, respectively (**P<0.01) in the 5-azacytidine-treated EC9706 cells. (B and C) Western blot analysis of the protein expression of CDH1 and SOX17. The expression levels of CDH1 (**P<0.01) and SOX17 (**P<0.01) was significantly upregulated by 5-azacytidine. 5-aza, 5-azacytidine.
Fig 3: 5-Azacytidine inhibits EC9706 cell proliferation via the upregulation of SRY-box containing gene 17 (SOX17). The downregulation of SOX17 by siRNA attenuatd the inhibitory effects of 5-azacytidine on cell proliferation. (A) Western blot analysis was used to detect the expression of SOX17 in the cells treated with 5-azacytidine and transfected with SOX17 siRNA. Compared with the DMSO vehicle control group, the protein expression of SOX17 was increased by 5-azacytidine treatment (**P<0.01). siRNA targeting SOX17 significantly attenuated the 5-azacytidine-induced upregulation of SOX17 (**P<0.01). (B) Cell counting kit-8 (CCK-8) assay of EC9706 cell viability. EC9706 cell viability was inhibited by 5-azacytidine treatment on days 3 and 4 (*P<0.05 and **P<0.01). SOX17 siRNA significantly impaired the effects of 5-azacytidine on days 3 and 4 (**P<0.01). (C) Cell colony-formation assay of EC9706 cells. 5-Azacytidine inhibited EC9706 cell colony formation, while SOX17 siRNA hampered the effects of 5-azacytidine. (D) Flow cytometry was carried out to examine the apoptosis of EC9706 cells. Compared with the negative control (DMSO-treated cells), 5-azacytidine promoted the apoptosis of EC9706 cells (**P<0.01). The 5-azacytidine-induced apoptosis was significantly inhibited by SOX17 siRNA (*P<0.05). 5-aza, 5-azacytidine.
Fig 4: Methylation of cadherin 1 (CDH1) and SRY-box containing gene 17 (SOX17) promoters is decreased by 5-azacytidine in EC9706. Methylation level analysis of CDH1 and SOX17 promoters. (A) Methylation analysis of CDH1 promoter in 5-azacytidine-treated EC9706 cells. Methylation level of the CDH1 promoter was decreased significantly by 5-azacytidine. (B) Methylation analysis of SOX17 promoter in 5-azacytidine-treated EC9706 cells. Methylation level of SOX17 promoter was decreased significantly by 5-azacytidine. 5-aza, 5-azacytidine.
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