(a) Inhibition of CPT-induced ATM phosphorylation at S794 and S1981 by roscovitine. Neurons differentiated from SH-SY5Y cells were pretreated with or without 10 μM roscovitine (Ros) for 30 min and followed with 10 μM CPT for the indicated periods of time. Phospho-S794, phospho-S1981 and total ATM were determined by immunoblotting. (b) Inhibition of CPT-induced ATM phosphorylation at S794 and S1981 by knocking down Cdk5. Neurons differentiated from SH-SY5Y cells were infected with control or Cdk5 RNAi adenovirus for 24 hours, and then treated with 10 μM CPT for the indicated periods of time. Cdk5, phospho-S794, phospho-S1981, total ATM and actin were determined by immunoblotting. (c) Inhibition of CPT-induced ATM activation and p53 phosphorylation by roscovitine. CGNs were treated with or without 10 μM Roscovitine for 30 min and then with 10 μM CPT treatment for 1 hour. ATM kinase activity, phospho-S15, total p53 and actin were measured in the same set of lysates. (d) Inhibition of CPT-induced ATM activation and p53 phosphorylation by knocking down Cdk5. CGNs were infected with Cdk5i or scrambled RNAi lentivirus for 72 hours and then treated with 10 μM CPT treatment for 1 hour. Cdk5 and ATM kinase activities and levels for Cdk5, phospho-S15, total p53 and actin were measured in the same set of lysates. (e) Reduction of CPT-induced γ-H2AX foci formation by roscovitine. CGNs were treated with or without 10 μM Roscovitine (Ros) for 30 min and then exposed to 10 μM CPT for 1 hour. γ-H2AX (green) was detected by immunocytochemistry, and nuclei were labeled with Hoechst (blue). The average numbers of foci/cell counted blindly are control, 0.26; CPT, 3.91; CPT+Ros, 1.08 (p< 0.001). (f) Reduction of CPT-induced γ-H2AX foci formation by knocking down Cdk5. CGNs were infected with CDK5 RNAi or control lentivirus for 72 hours and then exposed to 10 μM CPT for 1 hour. γ-H2AX (red) and GFP (green) were detected by immunocytochemistry, and nuclei were labeled with Hoechst (blue). The scale bars represent 10 μm. The average numbers of foci/cell counted blindly are control, 0.21; control+CPT, 3.00; Cdk5i, 0.13; Cdk5i+CPT, 1.22 (p< 0.001).