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. 2005 Sep 14;25(37):8349–8358. doi: 10.1523/JNEUROSCI.1570-05.2005

Figure 3.

Figure 3.

Cdk4 is required for Bim expression. A, B, Induction of Bim promoter-driven luciferase activity after NGF deprivation in cultures of sympathetic neurons (A) and neuronal PC12 cells (B) is blocked by inhibition/loss of cdk4 activity. Cultures were cotransfected with 0.5 μg of Bim–luc and 0.1 μgofthe Renilla luciferase expression construct pRL–CMV with 0.5 μg of empty pCMV vector (Control) or indicated construct, maintained for 24 h, and then deprived of NGF for 18 h, after which luciferase assays were performed. Data were normalized as in Figure 2 and are means ± SEM of three or four experiments. Asterisks denote statistically significant differences from control: A,*p < 0.004; B,*p < 0.001, ** p < 0.0003. C, Cdk4 and Bim siRNAs suppress cdk4 and Bim expression, respectively. HEK 293 cells were cotransfected with pCMS–EGFP expressing Flag–cdk4 or Flag–BimEL and pU6–cdk4–siRNA, pSIREN–Luc–siRNA, pSIREN–Bim–siRNA, or empty pU6 vector. After 48 h, cells were lysed and subjected to Western immunoblotting as described in Figure 1 A with anti-Flag and anti-EGFP as probes. D, E, Cdk4 siRNA reduces endogenous cdk4 expression in neuronal PC12 cells after NGF withdrawal. Neuronal PC12 cells were cotransfected with pSIREN–cdk4–siRNA (siCdk4) or pSIREN–Luc–siRNA (siLuc) and pCMS–EGFP, maintained for 48 h, and then deprived of NGF for 18 h, after which immunostaining with antibodies against cdk4 (red) and GFP (green) was performed. Percentage of stained cells pertains to the proportions of transfected cells (as indicated by staining for GFP) that show cdk4 staining either high (more than that of NGF-maintained cells) or low (equal or less than that of NGF-maintained cells). Data represent means ± SEM of three experiments. Approximately 50 cells were evaluated perculture. Asterisks denote statistically significant differences between low-staining cells and high-staining cells: *p < 0.0001. F, Cdk4 siRNA protects neuronal PC12 cells from death evoked by NGF deprivation. Cells were cotransfected with pCMS–EGFP and either empty pU6 (Control) or pU6–cdk4–siRNA and 24 h later subjected to NGF withdrawal (W/D). Numbers of transfected (green) cells were determined at indicated times, and percentage survival was calculated relative to the number of transfected cells present before exposure to apoptotic conditions. Data represent means ± SEM of three experiments performed in triplicate.