Fig. 6

Clemastine decreases SOD1 and stimulates autophagy via mTOR pathway in SOD1-G93A spinal microglia cultures. Spinal primary microglia from SOD1-G93A mice at PND 120 are exposed to clemastine 30 μM for 6 h. a Equal amounts of treated microglia lysates are subjected to western blotting with anti-SOD1 and anti-GAPDH. b Cells are analysed by means of a confocal microscope after staining with anti-CD11b (red) and anti-SOD1 (green) (scale bar 20 μm). c Equal amounts of treated microglia lysates are subjected to western blotting with anti-SQSTM1/p62 or in d with anti-mTOR and anti-p-mTOR. Anti-GAPDH was used for protein normalisation. e SOD1-G93A microglia are exposed to clemastine 30 μM and/or bafilomycin-A1 25nM for 6 h, and equal amounts of total lysates are subjected to western blotting with anti-LC3-I/II and anti-GAPDH for protein normalisation. f Cells are also analysed by means of a confocal microscope after staining with anti-LC3-I/II (red) (scale bar 20 μm). Total lysates of SOD1-G93A microglia exposed to clemastine and/or wortmannin 100 nM are subjected to western blotting with anti-SOD1 (g) or anti-CD68 (h). Anti-GAPDH is used for protein normalisation. Data represent mean ± S.E.M. of n = 3 independent experiments. Statistical significance is calculated by student’s t test referred to untreated cells (Ctrl), * p < 0.05 or to clemastine-treated cells, ^# p < 0.05