(a) The expression levels of Nur77 mRNA (top) and protein (bottom) in SMMC-7721 and Huh7 cells that were treated with ADC (10 μM) for indicated times. (b) Detection of methylation by methylation-specific PCR in clinical samples (top) and different liver cancer cell lines (bottom). U, unmethylation; M, methylation. (c) Snail and Nur77 expression levels detected in the same clinical HCC sample. Scale bars, 100 μm. (d) The negative correlation between the nuclear Snail and the protein levels of Nur77 in 82 clinical HCC samples. (e) Different reporters of Nur77 promoter, constructed as shown in Supplementary Fig. 6a, were transfected into Snail overexpression (top) or Snail knockdown (bottom) SMMC-7721 and Huh7 cell lines, and luciferase assays were perform to determine Nur77 promoter activity. (f) Overexpression of Snail (left) or knockdown of Snail (right) in SMMC-7721 and Huh7 cells affected expression levels of Nur77 gene (top) and protein (bottom). (g) The recruitment of Snail, HDAC1, HDAC2 or DNMT1 to Nur77 promoter revealed by ChIP assays in SMMC-7721 and Huh7 cells. (h) Snail-mediated deacetylation of H3K9 on Nur77 promoter in Huh7 cells detected by ChIP assays. Tubulin was used to indicate the amount of loading proteins. Data were represented as means±s.e.m. of at least three independent experiments. *P<0.05; **P<0.01; ***P<0.001. The data were analysed using one-way ANOVA followed by Tukey post hoc test in (e,f) and Pearson’s chi-squared test in (d).