Figure 4. Cdc25A knockdown sensitizes CGNs to excitotoxic death mediated by hypoxia and glutamate. CGN cultures were treated with varying concentrations of Cdc25 inhibitor NSC95397 and were subjected to 5 h of hypoxia in the absence of MK801. Neuronal survival was assessed following 1 h of reoxygenation. N = 3. A, B, CGNs treated with NSC95397 were subjected to 50 μm transient glutamate exposure for 70 min (A) and then and examined for survival 1 h later (B). N = 3. C, D, CGNs infected with AAV expressing shRNA to Cdc25A or control shRNA were treated with hypoxia without MK801 and assessed for survival. N = 3, p = 0.068 (Student's t test, two-tailed test; C) or transient glutamate exposure, N = 3. *p < 0.05 (Student's t test, two-tailed test; D). Neuronal survival was assessed by nuclei integrity following immunofluorescence staining with anti-GFP antibody and Hoechst stain. E–G, CGNs null for Cdc25B, N = 4 (E); Cdc25C: +/+, N = 3; −/−, N = 5; F) or doubly null for Cdc25B and Cdc25C, +/+, N = 5 and −/−, N = 5, *p < 0.5 (Student's t test, two tail; G) were subjected to transient glutamate treatment for 70 min and assessed for survival 1 h later. Neurons doubly null for Cdc25B and Cdc25C showed resistance to glutamate-mediated excitotoxic death. H, Western blot analysis showing increased Cdc25A protein level in Cdc25B and Cdc25C double-knock-out brain samples. I, Densitometry of Cdc25A protein as shown in H, N = 3, *p < 0.5 (Student's t test, two tail). J, Western blot analysis of Cdc25A protein levels following glutamate treatment for the indicated times. K, Quantitation of Cdc25A protein levels as in E. N = 3, p = 0.053 (Student's t test, one-tailed test). L, Cdc25A phosphatase activity following glutamate treatment for 30 min. N = 3.