Figure 5. KDM4A, but not KDM4B, binds with AR and suppresses AR target p27^kip1 by demethylating H3K4me3 in response to androgens in AN3CA cells.

A. AR expression in various EC cell lines; AN3CA cells were studied because of their low AR expression. B. AN3CA cells transiently transfected with mammalian expression vectors encoding AR or empty vector control were subject to co-IP using anti-AR or control antibodies before Western blotting using anti-KDM4B (B, left) or anti-KDM4A (B, right). C. KDM4A over-expression was confirmed by qRT-PCR in AN3CA cells. D. AN3CA cells were transiently transfected with either negative control (NC) or KDM4A expression plasmid (exKDM4A). Overexpression of KDM4A inhibited p27^kip1 mRNA expression in qRT-PCR assays in AN3CA cells E. the expression of other suppressor genes, including c-myc, did not affect p27^kip1 levels (D) *P < 0.05 compared with NC. ChIP for F. AR, G. KDM4A, H. H3K9me3, and I. H3K4me3 at the p27^kip1 promoter in AN3CA cells treated with DHT (100 nM) for 0, 60, or 180 min. *P < 0.05, **P < 0.01 compared with 0 min. J. AN3CA cells were transiently transfected with either negative control (NC) or KDM4A siRNA (siKDM4A) in steroid-depleted media for 72 h with or without 180 min of exposure to 100 nM DHT before ChIP with an anti-AR antibody K. an anti-H3K9me3 antibody L. or an anti- H3K4me3 antibody M. and subsequently analyzed at the p27^kip1 promoter. Nonspecific IgG was used as a negative control. N. AN3CA cells were subject to transient transfection with either negative control or KDM4A (siKDM4A) siRNAs in steroid-depleted media for 48 h before 24 h of exposure to 100nM DHT followed by protein extraction. AR, KDM4A, p27^kip1 (N) and global histone modifications O. were assessed with Western blots. Histone H3 and β-actin were used as loading controls.