Fig. 2

C/EBPβ enhancer hypomethylation increases C/EBPβ expression via induction of eRNA. a Detection of C/EBPβ eRNA by northern blot using both sense and antisense probes. b Correlation between C/EBPβ enhancer methylation and eRNA expression in eight liver cell lines denoted with Pearson correlation coefficient. C/EBPβ eRNA levels are ΔCt values using 18s rRNA as internal control. c Pyrosequencing analysis of C/EBPβ promoter and enhancer methylation in liver cell lines treated with or without 5-aza-dC. d qRT-PCR analyses of C/EBPβ eRNA and mRNA in liver cell lines treated with or without 5-aza-dC. e, f Knockdown of C/EBPβ eRNA by two independent siRNAs in liver cells downregulated (e) C/EBPβ eRNA and (f) mRNA expressions. g, h Knockdown of C/EBPβ eRNA abrogated (g) C/EBPβ eRNA and (h) mRNA reactivation after 5-aza-dC treatment. d–h C/EBPβ eRNA/mRNA levels were calculated by the 2^−ΔΔCt method using 18s rRNA as internal control, and are presented as fold-changes against the average values of the respective control groups (DMSO/siCtrl). i Correlation between C/EBPβ enhancer methylation and eRNA levels in human HCC tissues denoted with Pearson correlation coefficient. j Correlation between C/EBPβ eRNA and mRNA levels in human HCC tissues denoted with Pearson correlation coefficient. i, j C/EBPβ eRNA/mRNA levels are ΔCt values using 18s rRNA as internal control. Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 as calculated by Pearson correlation test (b, i–j), and unpaired two-tailed Student’s t-test (c–h)