Fig. 3
circRHOT1 regulates NR2F6 expression in HCC cells. a Bland-Altman plot for differently expressed genes (log2(Fold change) > 1.5) and seven selected genes were listed. Red plot denotes NR2F6. b The expression of the most downregulated genes was detected by qRT-PCR in WT and circRHOT1-deleted HCC cells. c The expression of NR2F6 was determined by western blotting. d The enrichment of circRHOT1 on the NR2F6 promoter was assessed by chromatin isolation by RNA purification (CHIRP) using specific biotin-labeled probes (5′-AACCTGCTGTCTTTGTCTGT-3′, 5′-GGAGGAACCTGCTGTCTTTG-3′ and 5′-CCCGGGGAGGAACCTGCTGT-3′) in HCC cells. Control probe sequence is 5′-ATTGTCCGATCGTCTCACGT-3′. e-g The enrichment of H3K27Ac (e), H3K27me3 (f) and RNA pol II (g) on the NR2F6 promoter was determined by chromatin immunoprecipitation (ChIP) in WT and circRHOT1-deleted HCC cells. h The promoter accessibility was evaluated by DNAse I digestion. i The binding region of circRHOT1 in the NR2F6 promoter was deleted, and then the enrichment of circRHOT1 was assessed by CHIRP. j The mRNA levels of NR2F6 were determined by qRT-PCR in the indicated HCC cells. **p < 0.01 and ***p < 0.001. All the data are representative of at least three independent experiments and presented as the means ± SD