Fig. 5.

CDKN2B-AS1 affects atherosclerosis by forming RNA-DNA triplex with CDKN2B promoter. a, RT-qPCR assay for the transcriptional expression of the CDKN2B gene following treatment with GSK343, an E2H2 inhibitor. b, RNA immunoprecipitation assay for the binding of EZH2 to CDKN2B-AS1 relative to IgG. c, RT-qPCR assay for the expression of CDKN2B mRNA. d, ChIP assay for the enrichment of CTCF in the CDKN2B promoter region relative to IgG. e, ChIP assay for the enrichment of EZH2 and H3K27me3 in the CDKN2B promoter region relative to IgG. f, ChIP assay for the enrichment of CTCF and H3K27me3 in CDKN2B promoter region relative to IgG, following treatment with GSK343, an E2H2 inhibitor. g The CDKN2B-AS1 TFO sequence predicted by LongTarget. h, The binding of CDKN2B-AS1 and CDKN2B promoter detected by in vitro triplex pull-down assay. i, RNA-DNA triplex formed by CDKN2B-AS1 and CDKN2B detected by EMSA. j, The binding of CDKN2B-AS1 and CDKN2B promoter detected by in vivo triplex capture assay. * p < 0.05 vs. the sh-NC, DMSO, oe-control oligo or dGTP group; ^#p < 0.05 vs. the oe-NC or GSK343 group; ^##p < 0.05 vs. the oe-CTCF group. The experiment was repeated 3 times independently. The t-test was used for comparison of data between two groups, and one-way ANOVA was used for comparison of data between multiple groups. sh-NC, cells transduced with pSIH1-H1-copGFP-sh-NC; sh-CDKN2B-AS1, cells transduced with pSIH1-H1-copGFP-sh-CDKN2B-AS1; oe-NC, cells transduced with LV5-GFP empty vector; oe-CTCF, cells transduced with LV5-GFP-CCCTC-binding factor; sh-CTCF, cells transduced with pSIH1-H1-copGFP-sh-CCCTC-binding factor.