FIG. 4.

Effects of DLC overexpression on differentiated PC12 cell apoptosis induced by NGF withdrawal. (A) Western blot analysis of PC12 cells stably expressing DLC. Lanes 1 and 2, PC-Off (parental) cells treated without (lane 1) or with (lane 2) doxycycline (10 μg/ml) for 48 h; lanes 3 and 4, PC-DLC cells stimulated without (lane 3) or with (lane 4) doxycycline (10 μg/ml) for 48 h. (B) Prevention of PC12 cell apoptosis induced by NGF withdrawal by DLC overexpression as determined by Hoechst staining. PC-Off and PC-DLC cells were differentiated by NGF for 7 days, followed by treatment with or without NGF withdrawal for 17 h. Data shown are the averages of three independent experiments, and error limits are within 3%. (C) Prevention of PC12 cell apoptosis induced by NGF withdrawal by DLC overexpression as determined by DNA fragmentation. Soluble cytoplasmic DNA from differentiated PC-Off or PC-DLC cells with (+) or without (−) NGF withdrawal for 17 h was analyzed by agarose gel electrophoresis. Some undegraded RNA was also strongly stained with ethidium bromide at the bottom of the gel. (D) Inhibition of caspase 3 activation induced by NGF withdrawal by DLC overexpression. Differentiated PC-Off or PC-DLC cells were incubated with or without anti-NGF antibody. At indicated time points, cells lysates (40 μg) were analyzed by Western blotting for caspase 3.