Fig. 4. Modulation of NGF-induced ceramide accumulation and PARP hydrolysis. (A) Modulation of ceramide accumulation by NGF. Five HH18 otic vesicles/per datapoint were labelled with 25 µCi/ml of [³H]palmitic acid for 24 hours as described in Materials and Methods. Otic vesicles were then carefully washed and incubated for 3 hours without additives (control) or with 4 nM NGF alone or in combination with 1 nM IGF-I, 2 µg/ml anti-NGF antibody, 9651 anti-p75^NTR antibody (1:100), 25 µM zVAD or 75 µM DEVD. Ceramide accumulation was measured as described in Materials and Methods. Results are expressed as mean±s.e.m. of at least five independent experiments performed in triplicate. Statistical significance estimated by ANOVA was as follows ^*P<0.005 versus control, #P<0.005 versus NGF. (B) NGF-induced cell death triggers PARP-hydrolysis. Hydrolysis of native poly-ADP-ribose polymerase (p116-PARP) produces a fragment of 85 kDa (p85-PARP). Quiescent HH18 otic vesicles (8 otic vesicles per data point) were incubated for 16 hours either in serum-free medium (Control) or in the presence of 4 nM NGF alone or in combination with 1 nM IGF-I, 2 µg/ml anti-NGF antibody, 9651 (1:100) anti-p75^NTR antibody, 25 µM zVAD or 75 µM DEVD. Incubation with the non-blocking p75^NTR antibody or agonist vehicles had no effect on PARP induction (data not shown). Otic vesicle lysates were analysed by western blotting with an anti-PARP antibody that recognizes both the intact and the proteolysis fragment. A representative blot of one of three independent experiments is shown. Densitometric quantification is shown as bars. Statistical significance estimated by ANOVA was as follows ^*P<0.005 versus control, #P<0.005 versus NGF. (C) C₂-Cer-induced cell death also triggers PARP-hydrolysis. Otic vesicles were incubated in serum-free medium (Control) or 5 µM C₂-Cer alone or in combination with 1 nM IGF-I, 25 µM zVAD or 75 µM DEVD. Otic vesicle lysates were analysed by western blotting with an anti-PARP antibody as above. A representative blot of one of three independent experiments is shown. Densitometric quantification is shown as bars. Statistical significance estimated by ANOVA was as follows ^*P<0.005 versus control, #P<0.005 versus C₂-Cer.