Fig. 7

HOXA5 recruited DNMT3A to increase the DNA methylation level in the promoter region of Slug. a. Correlation between Slug methylation and expression from the cBioPortal website. b. CpG island analysis of the promoter region of Slug and MeDIP primer design. TSS, transcription start site. c. MeDIP assay on 769P cells with vector/HOXA5-overexpression conditions. Primer A, B and C were specific to different region of the Slug promoter. d. Immunoprecipitation using Flag antibody in Flag-HOXA5 overexpression 769P cells. Non-specific IgG was used as negative control. e. Immunoprecipitation using DNMT3A antibody in Flag-HOXA5 overexpression 769P cells. Non-specific IgG was used as negative control. f. Immunofluorescence co-localization of HOXA5 and DNMT3A. DAPI was used to indicate the location of nuclei. g. ChIP-qPCR assay using a DNMT3A antibody on 769P cells with vector/HOXA5-overexpression conditions. Non-specific IgG was used as control. h. Immunoblotting of HOXA5-DNMT3A salvage experiment. 769P cells were transfected with DNMT3A siRNAs with or without Flag-HOXA5 overexpression. GAPDH was used as normalization control. i. Immunoblotting of HOXA5-DNMT3A salvage experiment. Caki-1 cells were transfected with DNMT3A siRNAs with or without Flag-HOXA5 overexpression. GAPDH was used as normalization control. j. Transwell assay of the 769P HOXA5-DNMT3A salvage experiment. Representative images (left) and quantification (right) data were showed. k. Transwell assay of the Caki-1 HOXA5-DNMT3A salvage experiment. Representative images (left) and quantification (right) data were showed. Data are mean ± SD, n = 3