Figure 5. ARHGAP27P1 epigenetically activated p15 and p16 transcription by binding to JMJD3. (A) FISH analysis of the subcellular location of ARHGAP27P1 in AGS and HGC-27 cells. (B) Subcellular fractionation and RT-qPCR analysis to determine the ARHGAP27P1 location. U6 and β-actin were used as nucleus and cytoplasm markers, respectively. (C) RIP experiments were performed in SGC-7901 cells with IgG, anti-JMJD3, EZH2, SMYD3, WDR5 or KMT2C antibody, and the coprecipitated RNA was subjected to RT-qPCR analysis for ARHGAP27P1. ARHGAP27P1 expression levels were presented as fold enrichment in diverse immunoprecipitates relative to that of IgG. (D) RIP assays were performed to determine the binding of ARHGAP27P1 to JMJD3 in SGC-7901 cells transfected with pcDNA-ARHGAP27P1, or cotransfected with pcDNA-ARHGAP27P1 and si-JMJD3. (E) ChIP-qPCR of JMJD3 occupancy and H3K27me3 binding to the p15 and p16 promoters in SGC-7901 cells, IgG as a negative control. (F) ARHGAP27P1-overexpressing SGC-7901 or AGS cells were cotransfected with si-JMJD3. The expression of p15, p16 and p57 was determined using RT-qPCR. (G) The protein levels of JMJD3, EZH2, SUZ12, H3K4me3, H3K27me3, p15, p16 and p57 were determined by Western blot. (H) Western blot analysis of JMJD3, EZH2, SUZ12, H3K4me3, H3K27me3, p15, p16 and p57 in HGC-27 cells transfected with si-ARHGAP27P1. *P < 0.05, **P < 0.01 versus vector or control. #P < 0.05 versus ARHGAP27P1.