Fig. 6

Stathmin 1/2 knockdown rescues microtubule and Golgi alterations triggered by mutant SOD1. a. Confocal images showing NSC-34 cells transfected with empty, SOD1^wt, SOD1^G85R or SOD1^G93A plasmids as well as ctrl, Stathmin-1 or Stathmin-2 siRNAs. Cells were immunolabelled for α-tubulin. Note that knockdown of Stathmin 1 or 2 rescues mutant SOD1-triggered microtubule alterations. Transfected cells were identified by expression of co-transfected MannII-GFP (not shown). Scale bar 5 μm. b. Diagram showing percentage of microtubule alterations (mean ± sd) under the different conditions. >150 cells were analyzed per condition. Statistical significance * p < 0.01 by Mann–Whitney test. Data are representative of three independent experiments. c. Confocal images showing NSC-34 cells transfected as in A and counterstained for GS28. Note rescue (arrowheads) of mutant SOD1-triggered Golgi fragmentation and GS28 dispersal by knockdown of Stathmin 1 or 2. Scale bar 5 μm. d-e. Diagram showing percentage (mean ± sd) of Golgi alterations labelled by MannII-GFP (d) or GS28 (e) under the different conditions. >150 cells were analyzed per condition. Statistical significance * p < 0.01 by Mann–Whitney test. Data are representative of four independent experiments. f-g. Western blots show efficient siRNA-mediated knockdown (KD) of Stathmin 1 (f) and Stathmin 2 (g) in NSC-34 cells transfected with empty, SOD1^wt, SOD1^G85R or SOD1^G93A plasmids as compared to ctrl siRNA (upper blots). Both Stathmin-1 or Stathmin-2 knockdown reduces the GS28 up-regulation triggered by SOD1^G85R or SOD1^G93A (middle blots). Loading control β-actin (lower blots) indicates reduced protein content in lanes 1 and 2 in F (empty ctrl, empty Stathmin-1 KD). The diagrams below show densitometric quantification of GS28 protein levels each normalized to β-actin (n = 4 blots, mean ± sd, * statistically significant differences between ctrl siRNA and Stathmin 1/2 siRNA as measured by Mann - Whitney test)