1. Effects of fAS stimulation on cathepsin-B activity, mitochondrial quality and microglial cell survival after autophagy inhibition.
Effects of fAS stimulation on cathepsin-B activity, mitochondrial quality and microglial cell survival after autophagy inhibition. (A,B) BV2 (A) or primary (B) microglial cells were left untreated or treated with spautin-1 (10 µM) for 24 h or FIP200 siRNA for 48 h. Cells were then stimulated with fAS (5 μM) for 24 h and cell death was evaluated using propidium iodide (PI) combined with Annexin V–FITC staining and subsequent flow cytometric analysis. Mean±s.e.m. percentages of Annexin V–FITC/IP double-positive dead cells are shown. (C) BV2 microglial cells were treated and stimulated as described in A and BCL-2, BCL-xL and cleaved caspase-3 protein levels were evaluated using flow cytometry. Graphs show representative histograms for each protein. (D,E) BV2 microglial cells were left untreated or treated with spautin-1 (SP-1, 10 µM) for 24 h or FIP200 siRNA for 48 h and stimulated with fAS (5 μM) for 24 h. Untreated (control) and scramble (scr) siRNA as controls also shown. Cathepsin-B activity (D) and mitochondrial mass and membrane potential (E) were measured as described in Fig. 5H and I, respectively. (F–H) BV2 cells were treated with the autophagy inhibitors spautin-1 (SP-1, 10 µM) for 24 h or FIP200 siRNA for 48 h (si) and stimulated with fAS (5 μM) at the indicated time points. (F) GAL-3 immunostaining. (G,H) Mean±s.e.m. quantification of GAL-3 puncta (G) and LysoTracker Red DND-99 staining (H). LysoTracker staining was evaluated using flow cytometry and results are relative to the mean fluorescence intensity (Gmean) of the control condition. Results were analysed by one-way ANOVA followed by post-hoc Dunnet's test; n=3. *P<0.05, **P<0.01, ***P<0.001; #P<0.05, ##P<0.01 when comparing fAS with fAS plus SP-1 or FIP200 siRNA treatment.
Claudio Bussi et al. J Cell Sci 2018;131:jcs226241
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