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. Author manuscript; available in PMC: 2022 Jan 7.
Published in final edited form as: Cancer Res. 2020 Aug 14;80(19):4212–4223. doi: 10.1158/0008-5472.CAN-20-1259

Figure 5. SUMOylation represses an EZH2 target gene CDH1.

Figure 5.

(A) Knockdown of SAE2 increased CDH1 gene promoter activity and overexpression of EZH2 reversed the effect. HCT116 cells were transfected with control siRNA (siCtrl) or SAE2-targeting siRNA (siSAE2) together with an empty vector (Ctrl) or EZH2-expression plasmid (EZH2). CDH1 promoter reporter plasmid and Renilla plasmid were also transfected and CDH1 promoter activity was measured using dual-luciferase assay after 72 h. (B) Stable knockdown of SAE2 increased CDH1 mRNA level and overexpression of EZH2 reversed the effect. (C) Knockdown of SAE2 increased and overexpression of EZH2 suppressed E-cadherin protein level. HCT116 cells without (−Dox) or with (+Dox) SAE2 knockdown were transfected with empty vector (Ctrl) or EZH2-expression plasmid (EZH2). CDH1 coding protein E-cadherin was detected using western blot. Relative protein band intensity was quantified using Image J, normalized to GAPDH and labeled below each blotting band. SAE2 level was blotted to confirm Dox-induced SAE2 knockdown and HA-tag was blotted to confirm the expression of transfected HA-EZH2. GAPDH was used as loading control. (D) Knockdown of SAE2 increased E-cadherin and ZO-1 but decreased N-cadherin, Snail, and Slug. Protein was extracted from HCT116 cells without (−Dox) or with (+Dox) SAE2 knockdown and SAE2, EZH2, E-cadherin, ZO-1, Snail, and Slug were detected using western blot. Relative protein band intensity was quantified using Image J, normalized to GAPDH, and labeled below each blotting band. (E) GSEA revealed that “Alonso_Metastasis_UP” (top) and “Bidus_Metastasis_UP” (middle) gene sets were enriched in the HT29 control group (shCtrl) in comparison to the SAE2 knockdown group (shSAE2), whereas “Bidus_Metastasis_DN” gene sets (bottom) were enriched in the SAE2 knockdown group compared to the control group. Estimated variation is indicated as SD, p values were derived using a two-tailed Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001.