Figure 5.

ANCR is required for EZH2 binding on PTEN promoter.

Notes: (A) RNA-binding protein immunoprecipitation (RIP) assay was performed in FLAG-EZH2 overexpressing SUNE-1 cell lines. Top, FLAG–EZH2 was immunoprecipitated by FLAG antibody and detected by Western blot assay. Bottom, ANCR expression level after RIP assay was detected by qRT-PCR. (B) ChIP analysis showed enrichment of EZH2, H3K27me3, RNA polII, and H3K4me3 on PTEN promoter in SUNE-1 cells transfected with siANCR1 or NC siRNA. IgG and 3′UTR region primer served as negative controls. (C) EZH2 protein levels in SUNE-1 cells transfected with siANCR1 or NC siRNA were detected by Western blot assay. GAPDH served as loading control. (D) EZH2 and H3K27me3 levels were detected by Western blot assay. GAPDH served as loading control. (E, F) PTEN mRNA levels and protein levels were detected by qRT-PCR and Western blot assay. Data were mean±SD of three independent experiments; Student’s t-test, *P<0.05 compared with indicated controls. The numbers above denote relative gray values of each band.

Abbreviations: ANCR, antidifferentiation noncoding RNA; ChIP, chromatin immunoprecipitation; qRT-PCR, quantitative real-time PCR.