Figure 5.

Activation of GSK-3β contributes to glutamate-induced cell death. (a) CTX TNA2 cells were treated with 8 mM glutamate for 0–48 h and cell lysates (30 μg/lane) were then analyzed using immunoblotting with anti-p-tyr216-GSK-3β, anti-p-Ser9-GSK-3β, and anti-t-GSK-3β antibodies. T-GSK-3β was used as an internal control to normalize the amount of proteins. (b–e) Cells were pretreated with 10–40 μM SB216763 for 1 h and then treated with glutamate for 48 h. (b) The morphology of cells was examined and the cells were photographed using a reverse-phase microscope. Scale bar, 100 μm. (c) The percentages of cells undergoing autophagy and apoptosis were measured as described in Materials and Methods. Cell viability and cytotoxicity were analyzed using the MTT assay (d) and the LDH release assay (e), respectively. (f and g) Cells were transfected with 100 nM control siRNA or GSK-3β siRNA for 48 h and then treated with 8 mM glutamate for another 48 h to determine the percentages of cells undergoing autophagy and apoptosis (f) and the cell viability (g). (h and i) Cells were transfected with 2 μg pcDNA3 or GSK-3β-S9A dominant active plasmid for 48 h and then treated with 8 mM glutamate for another 48 h to determine autophagy and apoptosis (h) and the cell viability (i). SB, SB216763; Glu, glutamate. Data are presented as mean±S.E.M. (n=3/group; 6/group in the MTT assay). *P<0.05, **P<0.01 versus respective controls. ^#P<0.05, ^##P<0.01 versus each respective glutamate group, one-way ANOVA