Figure 7. Elevation of adaptive stress responses enhances OL survival.

(a) OPCs from control and Tsc1cKO mutants at P5 were cultured in PDGFAA proliferation (Pro) or T3 differentiation media (Diff) for 24 h and stained with EthD-1 and Calcein AM. Corresponding upper and lower panels were taken in the same field. Scale bar, 50 μm. (b) The percentage of dying cells among OPCs isolated from control and Tsc1cKO mutants in proliferation or differentiation medium for 24 h. Data represent the mean±s.e.m. from three independent experiments; ***P<0.001, Student's t-test. (c–f) Primary OPCs from control and Tsc1cKO pups were cultured under (c,d) proliferation or (e,f) differentiation conditions for 24 h. The cell lysates were subject to western blot analysis using antibodies to p-PERK, p-eIF2α and eIF2α; α-tubulin: loading control. p-eIF2α and p-PERK levels were normalized to α-tubulin. The graphs in d and f depict the fold change of expression levels in Tsc1cKO over control cells (data represent the mean±s.e.m. from three independent experiments). *P<0.05; Student's t-test. (g) qRT–PCR of Gadd34 from control and Tsc1cKO OPCs under proliferation or differentiation conditions for 24 h. Data represent the mean±s.e.m from three independent experiments. *P<0.05; **P<0.01; Student's t-test. (h) Expression of Gadd34 and p-PERK in the spinal cord of control and Tsc1cKO mice at P14 was examined by western blot. α-Tubulin was used as loading control. (i) Primary OPCs from control and Tsc1cKO mice were treated with Gadd34 siRNA or scramble control siRNA 24 h and immunostained with antibodies to cl-Casp3 and Sox10. Scale bar, 25 μm. (j) qRT–PCR analysis of Gadd34 mRNA expression from OPCs transfected with Gadd34 siRNA or scramble control siRNA for 48 h. (k) Percentage of cl-Casp3⁺ Sox10⁺ cells from above-treated OPCs in i. Data represent the mean±s.e.m from three independent experiments. ***P<0.001; one-way analysis of variance test with Tukey's multiple-comparison test.