Research Paper Volume 14, Issue 13 pp 5523—5536

Figure 5. ZNF674-AS1 promoted methylation of CHST7 by recruiting EZH2. (A, B) qRT-PCR determined the distribution of ZNF674-AS1 in AGS/OXA and MKN-45/OXA cells. (C) The relative enrichment of ZNF674-AS1 immunoprecipitated by anti-EZH2 or anti-IgG in AGS/OXA and MKN-45/OXA cells was determined by RIP assay. (D, E) Biotin pull-down assay was performed to verify interaction between ZNF674-AS1 and EZH2, followed by qRT-PCR analysis to evaluate ZNF674-AS1 levels (D) and western blot analysis to examine EZH2 enrichment (E). (F) The relative enrichment of CHST7 promoter immunoprecipitated by anti-EZH2 or anti-IgG in AGS/OXA and MKN-45/OXA cells was assessed by ChIP-PCR assay. (G, H) The relative enrichment of CHST7 promoter immunoprecipitated by anti-EZH2 or anti-IgG in ZNF674-AS1-overexpressing or negative control AGS/OXA and MKN-45/OXA cells was evaluated by ChIP-PCR assay. (I, J) BSP analysis of the methylation levels of CHST7 promoter in AGS/OXA and MKN-45/OXA cells treated with pSin-vec+siNC, pSin-ZNF674-AS1+siNC, pSin-ZNF674-AS1+siEZH2 or pSin-ZNF674-AS1+ 5-Aza-CdR. Student’s t-test.