FIGURE 11.

Knockdown of p21 blocks JAZ-mediated neuroprotection. A, overexpression of p21 protects neurons from LK-induced death. CGNs transfected with GFP or p21-FLAG for 8 h were switched to either HK or LK medium for 24 h. Viability of transfected neurons was evaluated by immunocytochemistry as described under “Experimental Procedures.” Data represent the mean ± S.E. for four independent experiments. **, p < 0.01, p21-transfected neurons as compared with control (GFP-transfected neurons in LK). B, HT22 cell lines were transfected with pLKO1 (control) or three different p21 shRNAs named ShA, ShB, and ShC for 72 h. Cell lysates were collected, and Western blot analysis was performed using a p21 antibody. Tubulin serves as a loading control. C, CGNs were co-transfected with p21 shRNAs and either GFP (6.5:1) or JAZ-FLAG (5:2.5) for 48 h and then switched to HK or LK medium for 24 h. Immunocytochemistry was performed using GFP or FLAG antibody. Data represent the mean ± S.E. of four independent experiments. One-way analysis of variance was performed with Bonferroni's multiple comparison test. *, p < 0.05, GFP/ShB transfected in HK as compared with GFP/pLKO1-transfected neurons in HK. **, p < 0.01, GFP/ShC-transfected neurons in HK as compared with GFP/pLKO1-transfected neurons in HK. ##, p < 0.01, JAZ/ShB-transfected neurons in HK as compared with JAZ/FLAG-transfected neurons in HK. ###, p < 0.001, JAZ/ShC-transfected neurons in HK as compared with JAZ-FLAG-transfected neurons in HK.