Fig. 5: LINC00511 is required for the epigenetic repression of CDKN1B by interacting with EZH2.

a qRT-PCR analysis of the relative RNA expression levels after nuclear and cytoplasmic RNA separation. GAPDH was used as a cytoplasmic marker, and U1 was used as a nuclear marker. b Representative RNA-FISH images of the subcellular location of LINC00511 in UACC-812 and MDA-MB-231 cells (red). Nuclei were stained with DAPI (blue). 18S rRNA was used as a cytoplasmic marker, and U6 was used as a nuclear marker. c Analysis of the interaction probabilities of LINC00511 and EZH2 with the prediction software RPISeq. d RNA immunoprecipitation and qRT-PCR analysis of endogenous EZH2 binding to LINC00511 in UACC-812 and MDA-MB-231 cells with the anti-EZH2 antibody. IgG was used as the control. e Schematic of the RNA pull-down assays for the identification of LINC00511-associated proteins. f Western blot analysis of EZH2 following the pull-down of LINC00511 or antisense-LINC00511 in UACC-812 and MDA-MB-231 cells. HuR was used as a positive control. g qRT-PCR analysis of the EZH2 expression of UACC-812 cells and MDA-MB-231 cells following the knockdown of LINC00511 expression. h qRT-PCR analysis of the CDKN1B expression of UACC-812 and MDA-MB-231 cells following the knockdown of EZH2 expression. i Western blot analysis of the CDKN1B expression of UACC-812 and MDA-MB-231 cells following the knockdown of EZH2 expression. j ChIP and qRT-PCR analysis of EZH2 and H3K27me3 occupancy at the CDKN1B promoter region in UACC-812 and MDA-MB-231 cells following the knockdown of LINC00511 expression. IgG was used as a negative control. Enrichment was quantified relative to input controls. Data are shown as the mean ± SD. Student’s t test was used for the statistical analysis: *p < 0.05; **p < 0.01; ***p < 0.001. Data represent three independent experiments