Figure 3.

ATF4 protects PC12 cells against neuronal cell death induced by dopaminergic neuronal toxins. A, Neuronal PC12 cells were transfected with plasmids encoding shRNA against ATF4 (shATF4-A) or a scrambled sequence as a control (shCTRL) for 2 d and then treated with different concentrations of 6-OHDA for 20 h or MPP+ for 36 h. Survival was assessed by counting the number of viable GFP⁺ cells. B, shATF4-A recognizes the rat-ATF4 sequence but not human-ATF4. Plasmids encoding shATF4-A and either rat or human ATF4 cDNA (r-ATF4 and h-ATF4, respectively) were transfected into HEK293T cells, and cell lysates were blotted with ATF4 antibody. C, Neuronal PC12 cells were transfected with the indicated plasmids, treated with 6-OHDA and MPP+, followed by counting of GFP⁺ cells. D, Neuronal PC12 cells were transfected with plasmids encoding rat ATF4 cDNA and GFP (ATF4) or GFP alone (GFP). Cultures were treated with 6-OHDA for 20 h or MPP+ for 36 h, followed by counting of GFP⁺ cells. *p < 0.05, **p < 0.01, ***p < 0.001 by Student's t test for A and D and by ANOVA with Tukey's post hoc test for C. The results shown are from representative experiments, with each condition performed in triplicate. There were six, three, and five independent experiments performed for the studies shown in A, C, and D, respectively.