Fig. 6.

Inhibition of TBK1 suppresses NASH-associated p62 phosphorylation and protein inclusion without alleviating hepatosteatosis and liver damage. (A–E) Four-month-old C57BL/6 male mice kept on methionine-restricted choline-deficient (CD)-HFD for 2 months were subjected to daily administration of vehicle (Con, 5% Tween-80 and 5% PEG-400; n = 10) or BX795 (25 mg/kg body weight, i.p.; n = 5) for 10 days. LFD-kept mice (n = 5) of the same age were used as a negative control. (A) Livers were subjected to solubility fractionation. 1% Triton X-100-insoluble fractions were analyzed by immunoblotting (left panel) and immunoblot quantification (right panels). (B,D) Liver sections were subjected to p62 and F4/80 immunostaining, hematoxylin and eosin (H&E) staining, Oil Red O (ORO) staining and Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) staining. Hematoxylin counterstaining was used to visualize nuclei. Scale bars, 200 μm. Boxed areas are magnified in the insets. (C) Serum ALT levels were quantified. (E) Relative F4/80 expression was quantified through RT-PCR. All data are shown as mean ± s.e.m. NS, not statistically significant; *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t-test). Arrowheads indicate the exact or nearest position of the protein molecular weight markers (kD).