Fig. 4

Snail2 represses E-cadherin expression through histone methylation and deacetylation. a SW480-N and SW480-Snail2 cells express the luciferase reporter plasmid pGL3-E-cadherin-Luc. Bix01294, GSK343, and TSA were added to the cell growth medium and 24 h later, the luciferase activity was assayed and normalized to that of Renilla (pRL-SV40), which served as an internal control. Each data point represents the mean ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Experiments were performed twice in triplicate. b The level of H3K27me3 at the E-cadherin promoter in SW480-N and SW480-Snail2 was analyzed by ChIP. ChIP samples were also analyzed by quantitative real-time PCR (mean ± SD from three separate experiments; bottom panel). c LMK-235 and Tubastatin A were added to the cell’s growth medium, and 24 h later, the luciferase activity was assayed and normalized to that of Renilla (pRL-SV40), which served as an internal control. Each data point represents the mean ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. Experiments were performed twice in triplicate. d Co-immunoprecipitation assays were performed in SW480 cells transiently co-expressing plasmids encoding HA-tagged Snail2 and HDAC6 (upper), or HDAC6 and EZH2 (lower). Cell extracts were immunoprecipitated with HA antibodies or EZH2 antibodies, and bound HDAC6 was examined by western blotting. e GSK343 and TSA were added to the cell growth medium in SW480-Snail2 cell line and 48 h later, expression of E-cadherin and fibronectin in cells was analyzed by qPCR and shown as “relative mRNA levels.” Data represent the mean ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. f GSK343 and TSA were added to the cell growth medium in SW480-Snail2 cell line and 48 h later, expression of E-cadherin and fibronectin in cells was analyzed by western blotting. g The migratory ability of SW480-Snail2 cells treated or not with GSK 343, TSA, and Tubastatin A for 24 h was analyzed in wound healing assays. The statistical analysis is shown in the bar graph (mean ± SD from three independent experiments), and a representative experiment is shown in the right panel. Phase contrast images were taken at × 4 magnification