Fig. 3.

Ras regulates sympathetic neuron apoptosis.A, Quantitation of TUNEL-positive cells is shown. Cultures (n = 3) were infected with RasV12 (100 MOI) or N17Ras (200 MOI) in the presence of 10 ng/ml NGF. Twenty-four hours after infection, cultures infected with RasV12 were deprived of NGF, while those infected with N17Ras were maintained in 10 ng/ml NGF. Three days after infection, cells were processed for TUNEL and Hoechst staining. As controls, sympathetic neurons were infected with 200 MOI (control for N17 Ras) or 100 MOI (control for RasV12) of a recombinant adenovirus expressing GFP. Results are expressed as a percentage of TUNEL-positive neurons relative to the total number of Hoechst-labeled cells. B, C, Sympathetic neurons infected with N17Ras (200 MOI) and maintained in 10 ng/ml NGF for 3 d after infection were processed for TUNEL (B) and Hoechst staining (C). As determined by fluorescence microscopy, neurons that were TUNEL positive (B) had nuclei containing highly condensed or fragmented chromatin (C), confirming that they were undergoing apoptosis, whereas those that were TUNEL negative always had spherical and uniformly distributed chromatin.