Figure 6
Autophagy-induced TGF-β/Smad2/3 signaling drives mesenchymal transition of ductular cells from AAF/CCL4 livers. (A) Immunofluorescence images of Smad2/3 in ductular cells from normal and AAF/CCL4 livers. Nuclear localization was confirmed with DAPI staining. The percentage of Smad2/3 localized in the nucleus was determined by counting 50 immunofluorescence‐positive cells for each sample. (B) Cell culture supernatant of ductular cells transfected with control (Ctrl) or siRNA against ATG7 (siATG7) was subjected to ELISA detection for TGF-β1 levels. Data are expressed as mean ± SD from three independent experiments. (C) Representative Western blot analysis of phosphorylated (p)- and total Smad2/3 in ductular cells transfected with control or siATG7. The graph below shows the relative intensities of the p-Smad2/3 bands normalized to those for total Smad2/3 from three independent experiments (mean ± SD, n = 3). Full-length blots are presented in Supplementary Fig. S9. Statistical analysis was analyzed by Student’s unpaired t-test. *P < 0.05 compared with normal group; #P < 0.05 compared with AAF/CCL4 groups; +P < 0.05 compared with AAF/CCL4 with siATG7 group. (D) Immunoblots and quantification depicting expression of type I collagen, vimentin, α-SMA and ATG7 in ductular cells, which transfected with control or siATG7 for 72 hr, and subsequently treated with or without 20 ng/ml TGF-β1 for another 24 hr. Relative intensity (RI) shown was calculated by normalization of the intensities of each marker to the β-actin and to the value of normal cell. Full-length blots are presented in Supplementary Fig. S10.
