Fig. 5. IGF-I increases Akt phosphorylation and PCNA levels. (A) Modulation of NGF-induced ceramide accumulation by R59949. Ceramide accumulation was measured as described in Materials and Methods. Otic vesicles were incubated for 3 hours without additives (Control), 1 nM IGF-I, 1 µM R59949 or with 4 nM NGF alone or in combination with IGF-I and R59949. Statistical significance estimated by ANOVA was as follows ^*P<0.005 versus control, #P<0.005 versus NGF. (B) Quiescent HH18 otic vesicles were incubated for 30 minutes with 1 nM IGF-I, 4 nM NGF, 5 µM C₂-Cer, 25 µM Cer-1-P or combinations of these factors for the determination of Akt or for 24 hours to measure PCNA levels. When indicated, a 30 minute pre-treatment with 1 µM R59949 was performed. Otic vesicle lysates were analysed by western blotting for total Akt levels, for the active phosphorylated form of Akt (Ser473 P-Akt) and for PCNA levels. Representative blots are shown. The lower panel shows densitometric quantification values of activated Akt (Akt-P) referred to total Akt levels (grey bars) and of PCNA (dark bars). Control cultures were taken as 100. Values are given as mean±s.e.m. of independent experiments performed in duplicate. The statistical significance between incubations under different conditions estimated by ANOVA was as follows: ^*P<0.005 versus control, #P<0.001 versus IGF-I. R59949 plus IGF-I and NGF or C₂-Cer were statistically significant (P<0.001) with respect to their equivalent R59949-untreated conditions.