Fig. 5

MK5 upregulated SNAI1 expression by phosphorylating c-Jun. a. Immunoblotting was confirmed the protein levels of c-Jun, p-c-Jun(S63) and SNAI1 after MK5 overexpression and silencing in HCT116 and SW620 cells. b. Immunoblotting analysis of MK5, c-Jun, p-c-Jun(S63) and SNAI1 after MK5-AS1 overexpression and knockdown in HCT116 and SW620 cells. c. Coimmunoprecipitation was used to identify interaction between MK5 and c-Jun in HCT116 cells. d. After cotransfection with pcDNA3.1 MK5, si-c-Jun or control siRNA, the proteins levels of MK5, c-Jun, p-c-Jun (S63) and SNAI1 were determined by immunoblotting. e. Immunoblotting of MK5, c-Jun, p-c-Jun(S63) and SNAI1 proteins levels from pcDNA3.1 MK5, pcDNA3.1 c-Jun, pcDNA3.1 c-Jun (S63A) transfection samples treated with DMSO or c-Jun phosphorylation inhibitor SR11302 in HCT116 and SW620 cells. f. Dual luciferase reporter plasmids containing SNAI1 promoter or pGL3 basic were co-transfected into HCT116 cells with c-Jun or mutant c-Jun (S63A) in parallel. g. Dual luciferase reporter plasmids of SNAI1 promoter were designed to contain c-Jun binding sequences or not. h. Dual luciferase reporter plasmids containing SNAI1 promoter, SNAI1 promoter MUT or pGL3 basic were cotransfected into HCT116 cells with c-Jun plasmid in parallel. i. Identification of the c-Jun binding sequences in SNAI1 promoters by ChIP-PCR. Data were shown as mean ± SD for three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001