Fig 2: Quercetin alleviated steatosis and restored lipophagy in the liver induced by chronic alcohol. (a) Hematoxylin and eosin-staining of mice fixed liver tissue section, observed by light microscope (400×). The black arrow indicates LDs, the red arrow indicates inflammatory infiltration. (b–e) Serum ALT and AST, liver TG and serum TG. (f) The immunostaining of paraffin sections of mice liver for co-localization of LC3 and PLIN2. (g) LAMP2 and LC3 were observed by fluorescent microscope (400×). Representative images were shown. (h) Western blot analysis was performed to measure proteins of LC3 and P62 (n = 6). (i–k) GAPDH was used as a protein loading control, and the results were quantified in three independent experiments per condition, with densitometry analysis of (h). Values were shown as means ± SD (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001. CON: normal control group; E: ethanol group; EQ: ethanol plus quercetin group; Q: quercetin group.

Fig 3: Plin2 overexpression reverses ISCs phenotype via proliferation and fibrogenesis signaling pathways. (a) Representative photomicrographs of phenotype change in activated ISCs transduced with Plin2 gene overexpressed or NC. (b) The protein expression of α-SMA, Col I, and FN in ISCs overexpressed Plin2 was detected by western blotting. (c) The lipid accumulation observation was measured in ISCs overexpressed Plin2. (d) The protein expression of Smad3-TGF-β in ISCs overexpressed Plin2 was detected by western blotting. Magnification: 40x; scale bars: 50 μm. ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. Error bars shown as ± SE of three independent repeated experiments.

Fig 4: Biological function of HBx on cell viability, cytoskeleton remodeling and lipid metabolism in vitro(A) The HBx promotes the Huh-7 cells proliferation compared with the control group (*p < 0.05). (B) The total and free intercellular cholesterol concentration of Huh-7/myc-HBx cell were significantly increased compared with control (*p < 0.05). (C) The lipid droplet was accumulated in Huh-7/myc-HBx cells using the oil red O staining. (D) The ADFP mRNA level of Huh-7/myc-HBx cells was significantly increased compared with control group (*p < 0.05). (E) The cytoskeleton and lipid metabolism related proteins, including CDC42, CFL1, PPARγ and ADFP in Huh-7/myc-HBx cells were significantly increased compared with control group. (F) The lipid droplet was obviously accumulated in 12M and 24M p21HBx/+ mice using the oil red O staining.

Fig 5: Validation for the amount changed protein CDC42, CFL1, ADFP and PPARγ in p21HBx/+mice and WT littermates by WB and IHC(A) Quantitative MS analysis of CDC42 abundance in 12M and 24M p21HBx/+ mice and their corresponding WT littermates. (A1) MS spectra and monoisotopic m/z values of a detected peptide from CDC42 protein are shown. (A2) WB analysis of CDC42 in 12M and 24M p21HBx/+ mice. GAPDH was used as the loading control. The experiment was repeated three times with similar results. (A3) Quantitative analysis of WB result for CDC42 using GAPDH as a control. Asterisk represents significant difference from control. (A4) IHC analysis of CDC42 in livers of 12M and 24M p21HBx/+ mice. Proteins of CFL1 (B), SEPT9 (C), ADFP (D), and PPARγ (E) in 12M and 24M p21HBx/+ mice and their WT littermates were analyzed by WB (upper panels) and IHC (lower panels). GAPDH was used as a loading control in WB.