Effects of AntiOxCIN4on hepatic lipid content and composition of WD-fed mice and FFAs-treated human HepG2 cells. (A) Hepatic lipid accumulation. Lipid quantification was obtained from three independent images/per animal of each experimental group of H&E staining. (B) Representative image of hepatic neutral lipid profile in WD-fed mice in the absence/presence of AntiOxCIN4 (2.5 mg/day/animal) using thin-layer chromatography (TLC). Several parameters were evaluated: triglycerides (TGs), diacylglycerols (DAGs), cholesteryl esters (CEs) and free cholesterol (chol). (C) Fatty acyl chain composition of hepatic triglycerides in terms of saturated fatty acids, palmitoleate, oleate, linoleate and ω-3 fatty acids in liver homogenates from WD-fed mice in the absence/presence of AntiOxCIN4 (2.5 mg/day/animal). (D) Neutral lipid accumulation (upper) and respective cell mass (lower) in human HepG2 cells treated with vehicle (BSA) or FFAs (24 h, 250 μM) in the absence/presence of AntiOxCIN4 (48 h, 100 μM). (E) Typical background-corrected images of HepG2 cells stained with the LipidTOX Green (lipids, green) and Hoechst 33342 (nucleus, blue), and treated with vehicle (BSA) or FFAs (24 h, 250 μM) in the absence/presence of AntiOxCIN4 (48 h, 100 μM) (upper). The LipidTOX Green and Hoechst 33342 fluorescence intensity was color-coded to green and blue, respectively. Average lipid droplet number, area and size was calculated from four images in multiple experiments (lower). (F) Triglycerides (left) and intracellular fatty acids (right) in cells treated with vehicle (BSA) or FFAs (24 h, 250 μM) in the absence/presence of AntiOxCIN4 (48 h, 100 μM). Data are expressed as the mean ± SEM (N = 5 per cage for the in vivo study and N = 4 for the HepG2 studies) and the results were normalized to the respective control condition (set as 100%). Statistically significant compared using two-way ANOVA followed by Fisher's LSD test for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.0005, ****P < 0.0001 vs SD or Vehicle + BSA); (#P < 0.05, ##P < 0.01 vs WD or Vehicle + FFAs).