Fig. 4

Autophagy activation by SFN was critical in LPS-induced primary GMECs. Cells were pretreated with SFN (5 μmol/L) for 12 h in the absence or presence of LPS (10 μg/mL) for 6 h. A Autophagy assay kit was used to detect autophagosome (Bar = 50 μm). B Fluorescence intensity analysis of autophagy shown in A, n = 30 randomly selected cells. C Autophagosome detection by transmission electron microscopy. Arrows indicate the autophagosome. D and E Western blot analysis of LC3 protein (n = 3). F Immunofluorescent analysis of LC3 puncta (green) and nuclear staining with DAPI (blue) in GMECs pretreated with Baf-A1 (10 μmol/L) for 1 h in advance and then treated with SFN (5 μmol/L) for 12 h in the absence or presence of LPS (10 μg/mL) for 6 h (Bar = 20 μm, n = 3). One-way ANOVA, Dunnett's post-hoc test. Data are presented as the mean ± SEM. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 and ^****P < 0.0001