Fig. 6: DHA inhibits proteins involved in the polycomb repressive complex.

a Immunoblot analysis of protein extracts obtained from DU145 and PC-3 treated with DHA (5 μM) using anti-phospho EZH2, and anti-JARID2, anti-EZH2 and anti-GAPDH. b DHA Treatment of PCa cells inhibits JARID2-EZH2 interaction. DU145 cells were treated with 5 µM of DHA and DMSO (0.05%) and protein extracts were immunoprecipitated using anti-JARID2 or anti-EZH2 antibodies. The interaction between the proteins was detected by anti-EZH2 (right) or anti-JARID2 antibodies. c Inhibition of JARID2 protein regulates miR-34a and miR-7 expression and Axl expression. RT-PCR analysis of miR-34a, miR-7, and Axl expression levels of in DU145 cells lacking JARID2 expression. DU145 were transfected with JARID2 siRNA or GFP siRNA duplexes (50 nM). Total RNA was collected and was analyzed 24 h post transfection. ΔCt values graphed are relative to the endogenous control RNU6B small RNA (for miR-34a and miR-7) and GAPDH for Axl. Data are representative of three independent experiments and all values shown are mean ± SD from a representative experiment; *p < 0.05, two-tailed Student’s t-test. d ChIP-qPCR analysis demonstrating the effect of DHA treatment on DU145 and C4-2 cells on H3K27me3 at specific gene loci. PCa cell lines were treated with 5 uM of DHA or DMSO and subjected to ChIP analysis using antibodies against H3K27me3 or control IgG. The signals of H3K27me3 at the indicated genomic locations were normalized to that obtained from IgG ChIP at the same location to calculate their enriched signal. e Schematic representation of DHA inhibition of Axl expression via regulation of microRNAs and proteins of the polycomb repressive complex 2