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. 2016 Jun 30;7:11960. doi: 10.1038/ncomms11960

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(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).