Figure 4

JMJD2A binds to ARHI gene promoter and represses ARHI transcriptional activity. (A, B) Western blot and quantitative real-time PCR (qPCR) analysis of expressions of JMJD2A and ARHI in breast cancer cell line MDA-MB-231. siRNA against JMJD2A and JMJD2A expression plasmid were used, respectively. ARHI expression level was upregulated in the siRNA group and downregulated in the JMJD2A group. (C, D) Western blot and qPCR analysis of expressions of JMJD2A and ARHI in breast cancer cell line MCF-7. The results were proved similar to those in breast cancer cell line MDA-MB-231. (E) JMJD2A binds to the ARHI gene promoter with two binding sites (A1 and A2). Anti-JMJD2A antibody was adopted. Normal IgG was used as negative control. The A1 site is critical for histone deacetylase (HDAC) binding with the ARHI promoter while A2 for E2Fs binding with ARHI promoter. GAPDH was used as a loading control. Two binding sites were both verified. *P <0.05, **P <0.01. ARHI, Aplasia Ras homolog member I; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.