Fig. 7.

Mitochondrial H₂O₂ production and reduction in GSH/GSSG ratio trigger autophagy activation and LD disappearance during HSC activation in vitro. Freshly isolated HSCs were cultured for 7 days. (A) Cells (Day 0 and Day 5) were treated with 10 mM MitoPY1 for 60 min at 37 °C. The medium was exchanged for fresh media containing DAPI (1.5 ml; 10 mg/ml stock solution)· H₂O₂ (1 mM) was used as a positive control. Na Py (10 mM) was pretreated for 1 h. After the treatment, Fluorescence of MitoPY1 (green), and DAPI (blue) was detected by fluorescent microscopy, (B) LC3-II conversion was determined by Western blot analysis, (C) the expression of P62 was detected by Western blot analysis. (D) Cells were pretreated with Cat (5000 Units) and SOD (1000 Units) for 1 h. Following the treatment, the expression of LC3-II was assessed by immunofluorescence. (E) The GSH/GSSG ratio was measured in freshly isolated HSCs cultured for 7 days. Besides, Cells (Day 0 and Day 5) were pretreated with Na Py (10 mM) or Cat (5000 Units) for 1 h. Following the treatment, the GSH/GSSG ratio was measured. For the statistics of each panel in this figure, data are expressed as mean±SD (n=3); *P<0.05 versus control, **P<0.01 versus control, ***P<0.001 versus control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)