FIGURE 3.

Antioxidants reduce phosphorylation of c-Abl and prevent motoneuron death induced by ACM-SOD1^G93A. (A) Flow diagram of experiment. ACM-SOD1^G93A was applied to 4 DIV spinal cord cultures acutely (for 90 min when c-Abl-P peaks; see Figure 1) or chronically (3 days), either alone or in the presence of antioxidants Trolox (1 μM), esculetin. (25 μM), or tiron (25 μM) Phosphorylation of c-Abl was measured at 4 and 7 DIV. Cell survival was measured at 7 DIV. (B) Graphs showing fluorescence intensities (RIU) for c-Abl-P at 4 DIV when treated acutely (90 min) with ACM-SOD1^G93A alone or ACM-SOD1^G93A plus the antioxidants; motoneurons (B₁), interneurons (B₂) and glial cells (B₃) were identified by immunostaining (as in Figure 1). (C) Same as in (B), but c-Abl-P is measured at 7 DIV when treated chronically (3 days) with ACM-SOD1^G93A alone or in the presence of the antioxidants. (D,E) Graphs showing the relative percentage of motoneurons that survived at 7 DIV, after being treated with antioxidants and ACM-SOD1^G93A (D) or ACM-SOD1^WT (E). Values represent mean ± SEM from at least 3 independent experiments performed in duplicate, analyzed by One-Way ANOVA followed by a Tukey post hoc test. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 relative to control conditions. #p < 0.05, ##p < 0.01, ###p < 0.001 compared to survival with the ALS-causing ACM to at 7 DIV without antioxidants.