Fig. 4

Identification Slug as a target of SMYD3-ANKHD1 in HCC. a Real-time PCR was performed to detect the mRNA expression of SMYD3-dependent target genes and EMT- inducing transcription factors in huh7 cells stably overexpressing SMYD3 with or without ANKHD1 knockdown. b Western blot detection of the expression of E-cadherin and Slug in huh7 cells stably overexpressing SMYD3 with or without ANKHD1 knockdown. c Two putative SMYD3 binding sites in human SLUG gene promoter region. d Luciferase reporter assay of huh7 cells cotransfected with the indicated luciferase reporter (wide type SLUG gene promoter-luciferase construct or its SMYD3 binding site mutants) and the empty vector or SMYD3 expression vector. e Nuclear extracts prepared from cells treated with pCDNA3.1-SMYD3 or pCDNA3.1-control were incubated with the biotin-labeled oligonucleotides probe corresponding to binding site 1 in the SLUG gene promoter to perform EMSA. Different fold excess of unlabeled oligonucleotides probe for binding site 1 were used to compete with the interaction between the labeled probe and SMYD3. f-h ChIP assays were performed in huh7-control and huh7-SMYD3 stable cell lines using antibodies against SMYD3, H3K4me3, and H3K9, K14Ac; immunoprecipitated DNA was measured by real-time PCR using primers for amplifying the SMYD3-binding regions in the SLUG gene promoter. Data are shown as mean ± SD of three separate experiments. *P < 0.05