Figure 5. ATRA upregulated HOXB13 expression by inhibiting EZH2 and DNMT3b in DU145 cells.

Treatment of 80 µM of ATRA upregulated HOXB13 and downregulated EZH2 and DNMT3b expressions at mRNA level (A) and protein level (B) in DU145 cells. 5-aza-dc was used as a positive demethylation control. Control 1 was isopycnic absolute ethanol and control 2 was isopycnic acetic acid (C) ATRA impaired the bindings of EZH2 and DNMT3b, and reduced the H3K27me3 level at HOXB13 promoter. ChIP assays were performed in DU145 cells treated with 80 µM ATRA and immunoprecipitated with anti-H3K27me3, anti-DNMT3b or anti-EZH2 antibody. The amounts of precipitated endogenous HOXB13 promoter DNA were determined by PCR. (D) BSP assays showed the reduced methylation of HOXB13 promoter in DU145 cells after treatment upon 80 µM ATRA, compared with the untreated control.