Fig. 5

Stathmin 1/2 overexpression phenocopies expression of mutant SOD1. a. Images showing microtubule alterations in NSC-34 motor neurons transfected with Myc-tagged Stathmin 1 or Stathmin 2, as compared to control cells. Cells were cultured for 2 DIV, fixed with paraformaldehyde and analyzed with antibodies α-tubulin (upper panels), detyrosinated tubulin (lower panels) or Myc (not shown). Scale bar 5 μm. b. Quantitative analyses showing percentage of cells with rarefied or broken microtubules (α-tubulin or detyrosinated tubulin). Data represent one typical out of five independent experiments each done in quadruplicate per condition. Number of cells analyzed (ctrl, Stathmin 1, Stathmin 2): α-tubulin (175, 174, 139), detyr-tubulin (184, 146, 152). Statistical significance by Mann–Whitney test: * p < 0.001 as compared to mock. c. Images showing Golgi alterations (MannII-GFP, GS28) together with microtubule alterations (α-tubulin) in NSC-34 motor neurons transfected with Stathmin 1 or Stathmin 2 as compared to control cells. Scale bar 5 μm. d. Quantitative analyses showing percentage of cells with Golgi alterations (MannII-GFP) or GS28 dispersal). Data represent one typical out of five independent experiments each done in quadruplicate per condition. Number of cells analyzed (ctrl, Stathmin 1, Stathmin 2): MannII-GFP (>500 each). GS28 (482, 434, 526). Statistical significance by Mann–Whitney test: * p < 0.001 as compared to mock. e. Western blot analysis of cells transfected with plasmids encoding Myc-tagged Stathmin 1 or Stathmin 2 or with an empty control plasmid (pcDNA). GS15 and GS28 levels are increased after overexpression of Stathmin 1 by 2.7 ± 0.3 and 2.6 ± 0.3 (mean ± sd) respectively. A similar increase in Golgi SNAREs is observed after overexpression of Stathmin 2 (GS15 2.6 ± 0.2, GS28 2.9 ± 0.3). n = 4 blots corresponding to independent experiments, * p < 0.01 by Mann - Whitney test