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Fig. 7. Effects of PI 3-kinase inhibition on OBSC proliferation, cell death and OBSC number. Cultures of OBSCs derived from E14.5 embryos were plated for 48 hours in conditions of proliferation, in the presence or absence of 100 ng/ml IGF-I, and they were treated with 30 µM LY294002 for the last 24 hours. (A) P-Akt^Ser473 phosphorylation was strongly inhibited by LY294002 while the reduction of P-Akt^Thr308 was moderate, without any change in the levels of P-Erk1/2. One representative immunoblot out of three experiments is shown. (B,E,F) Proliferating cultures were incubated with 5 µM BrdU for 22 hours before fixation. The addition of LY294002 significantly decreased the percentages of BrdU-positive cells in the presence of BSA or IGF-I. (C) LY294002 caused a significant reduction in the total number of cells growing in IGF-I and augmented the percentage of TUNEL-positive cells in the presence of BSA or IGF-I (D,G,H). Results are the average ± s.e.m. from 4-6 cultures. Statistical analysis was performed using Student's t test. (*P<0.05 **P<0.01.) Bar, 30 µm.

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