Fig. 4.

Combined effect of NSA with the D-FAT cocktail on defatting in fat-loaded PHH. (A-F) Normal human hepatocytes in primary culture were incubated with or without FFA mixture (OA:PA, 500:250 µmol/l) for 48 h, and thereafter FFA-loaded PHH were treated with D-FAT, D-FAT+NSA or vehicle for 24 h. (A) Quantification of lipid droplet content, assessed by Oil Red O staining. Left panel shows quantification of Oil Red O staining, normalized for the number of DAPI-stained nuclei; right panels show representative images (lower panels show magnification of boxed areas in upper panels). (B) Quantification of intracellular TG content normalized for cell protein. (C,D) RT-qPCR analyses of genes involved in fatty acid β-oxidation (C) (CPT1A, PGC1A, ACOX1) and lipogenesis (D) (SREBP1, FAS). (E,F) Genes involved in autophagy (LC3, SIRT1) were examined by RT-qPCR analyses (E) and western blotting (F). Data are mean±s.e.m. of six cell preparations, shown relative to the vehicle. *P<0.05 versus vehicle (one-way ANOVA). Scale bars: 50 µm.