Fig 2: ACSS1/2 positively correlates with histone acetylation and FASN expression in human hepatocellular carcinoma.(a) FASN, ACSS1 and ACSS2 protein levels (upper panels, normalized against ß-actin) and H3K9, H3K14, H3K18, H3K23, H3K27 and H3K56 acetylation levels (lower panels, normalized against histone H3) in 53 pairs of HCC (each paired with cancerous tissue (designated as C) and adjacent normal tissue (designated as N)) were analysed by western blot. Two pairs of representative samples were shown. For the other 51 pairs of samples, please refer to Supplementary Fig. 6b. (b) Heatmap of protein expression (ACSS1, ACSS2 and FASN) and histone acetylation levels (H3K9ac, H3K14ac, H3K18ac, H3K23ac, H3K27ac and H3K56ac) in all 53 pairs of HCC. Data were presented as Z-score of relative protein expression or histone acetylation. Tumours with 1.5-fold higher expression of ACSS1 or ACSS2 or both than that of adjacent normal control tissue are grouped into ACSS-high tumours (tumour/normal =1.5, n=26), while ACSS-low tumours (tumor/normal<1.5, n=27) express both ACSS proteins at 1.5-fold lower than its normal control. (c) H3K9ac (P=0.0220), H3K14ac (P=0.0289), H3K27ac (P=0.0034), and H3K56ac (P=0.0410), are significantly stronger in ACSS-high tumours than that in ACSS-low tumours. Statistical analyses were performed with a two-tailed unpaired Student's t-test (*P<0.05; **P<0.01; NS, not significant). (d) FASN protein level is significantly upregulated in ACSS-high tumours (P=0.002). Statistical analyses were performed with a two-tailed unpaired Student's t-test (**P<0.01). (e) Representative immunohistochemical staining results for ACSS1, ACSS2, FASN and H3K9/K27/K56 acetylation in adjacent normal tissue (N) and hepatocellular carcinoma (C). Scale bar, 100 µm.

Fig 3: Both ACSS1 and ACSS2 are involved in acetate-induced epigenetic regulation of de novo lipogenesis.(a) Stably concurrent knockdown of ACSS1 and ACSS2 totally blocks acetate-induced increase of histone acetylation levels. Histone acetylation levels in shscr or shACSS1/2 HepG2 cells treated with or without 2.5 mM acetate under normoxia or hypoxia were analysed by western blot. (b) 13C2-labelled H3K9ac and H3K27ac levels are decreased by ACSS1/2 knockdown. Quantification of 13C2-labelled histone H3 acetylation levels in shscr or shACSS1/2 HepG2 stable cell line treated with [U-13C2]-acetate for 4 h under hypoxia was analysed by LC-MRM MS. The percentage indicates the ratio of H3K[13C2-ac]/total H3Kac in each site. The results were presented as mean±s.d. of triplicate experiments (*P<0.05; ***P<0.001; by Student's t-test). (c,d) ACSS1/2 knockdown totally blocks acetate-induced histone acetylation association with FASN and ACACA promoter. ChIP-qPCR assays were performed to determine H3K9, H3K27 and H3K56 acetylation enrichment at FASN (c) and ACACA (d) promoter region in shscr or shACSS1/2 HepG2 stable cell lines treated with or without acetate under hypoxia for 4 h. IgG was included as a negative control. The results were presented as mean±s.d. of triplicate experiments (**P<0.01; ***P<0.001; NS, not significant; by Student's t-test). (e) ChIP-qPCR assays were performed to determine H3K9, H3K27 and H3K56 acetylation enrichment at ACLY promoter region in shscr or shACSS1/2 HepG2 cells treated as in c. IgG was included as a negative control. The results were presented as mean±s.d. of triplicate experiments (NS, not significant; by Student's t-test).