Figure 5.

The activation of autophagy is associated with DHA-induced GATA6 accumulation and HSC senescence. Activated HSCs were treated with DHA at various concentrations for 24 h or DHA at 20 μM for various hours. (A) Western blot and densitometric analysis were used to show the response of autophagic regulators in activated HSCs. (B) Real-time PCR and western blot data were used to show levels of different autophagy related genes in activated HSCs treated with DHA (5, 10 and 20 μM) or Rapa (100 nM) for 24 h compared with versus vehicles treated cells (Control). (C) Activated HSCs were transfected with a plasmid containing LC3-II tagged at the N terminus with green (GFP) and red fluorescent protein (RFP). This probe allows distinction of early autophagosomes (overlapping GFP+RFP puncta generating yellow puncta on overlay) and late autolysosomes (RFP puncta), as GFP fluorescence is quenched in the acidic autolysosomes. Images were examined under a × 20 lens on an Olympus fluorescence microscope using standard filter sets for GFP and RFP. (D) Primary HSCs were treated with DMSO or 20 μM DHA following 20 ng/ml PDGF-BB treatment for indicated hours. Longevity protein degradation was detected by LC–MS/MS. (E) Transmission electron microscope (TEM) analysis was used to show increased autophagosomes and autolysosomes in HSCs treated with 20 nM PDGF-BB and 20 μM DHA for 24 h compared with versus vehicles treated cells (Control); For the statistics of each panel in this figure, data are expressed as mean±S.D. (n=3); *P<0.05 versus control, **P<0.01 versus control, ***P<0.001 versus control