FIGURE 7.

The cytoprotective effect of Nes-S in N2a neuroblastoma cells. A, promitotic function of Nes-S, as revealed by time-course MTT assays of N2a/NestS and N2a/vector cells. These cells were passaged 12 h before being cultured in either serum-containing (S) or serum-free (SF) medium. The time of medium change was designated as 0 h. The cell number at each time point was normalized to that of N2a/vector cells cultured in serum-containing medium at 0 h. The N2a/NestS cells showed an ∼2-fold proliferation rate when cultured in serum-containing medium, as compared with N2a/vector cells (n = 4, **, p < 0.01, two-tailed t test). Both cell types did not proliferate in serum-free medium. Error bars indicate S.E. B, prosurvival function of Nes-S, as determined by MTT assays of N2a/NestS and N2a/vector cells, as well as untransfected N2a cells (N2a/wt). The survival rates of N2a/NestS cells upon 250 and 325 μm H₂O₂ treatments (95.8 ± 5.3 and 64.5 ± 6.0%, respectively) were significantly higher than those of N2a/vector cells (56.7 ± 4.2 and 23.2 ± 5.9%, respectively) and N2a/wt cells (44.9 ± 1.7 and 19.4 ± 6.8%, respectively). There was no significant difference in survival rates between N2a/vector cells and N2a/wt cells (n = 4, **, p < 0.01, ***, p < 0.001, two-tailed t test). Error bars indicate S.E. C, caspase-3 activation of N2a/NestS and N2a/vector cells. N2a cells transiently expressing either pEGFP (N2a/vector) or pEGFP-NestS (N2a/NestS) were subjected to serum starvation for 12 h and treated with 250 μm H₂O₂, and their whole cell extractions were collected at various time points. The results showed that expression of EGFP-tagged Nes-S protein attenuated the activation of caspase-3. D, co-immunoprecipitation of Cdk5 and EGFP-tagged Nes-S protein. N2a cells transiently expressing either pEGFP (N2a/vector) or pEGFP-NestS (N2a/NestS) were subjected to serum starvation for 24 h before harvesting. The lysates were immunoprecipitated with anti-GFP and immunoblotted (WB) with anti-Cdk5. Total lysates were also immunoblotted with anti-Cdk5 as a positive control. Cdk5 was detected in the immunoprecipitates (IP) from N2a/NestS lysates, but not in that from N2a/vector. Arrowhead, Cdk5. Asterisks, IgG bands. E, effect of Thr-316 point mutation on cytoprotective function of Nes-S. The viability of N2a/NestS-T316A cells was significantly higher than that of N2a/NestS cells (87.5 ± 2.3 and 64.5 ± 6.0%, respectively). The viability of N2a/NestS-T316D cells was significantly lower than that of N2a/NestS cells (36.5 ± 6.4 and 64.5 ± 6.0%, respectively). No significant difference in survival rates was observed among N2a/NestS-T316D, N2a/vector, and N2a/wt cells (n = 4, *, p < 0.05, **, p < 0.01, two-tailed t test). Error bars indicate S.E. UT, untreated.