Fig. 4. PTEN overexpression reduces the differentiation of OBSCs into neurons and astrocytes. OBSC cultures were infected in parallel with a vector expressing PTEN-GFP or with a vector expressing GFP alone. Three days later, the cells were passaged and cultured for 2-3 days in conditions of proliferation (with EGF and FGF-2) or differentiation (in the absence of mitogens), in the presence or absence of insulin/IGF-I (10 µg/ml of insulin had similar potency as 100 ng/ml IGF-I to stimulate OBSC differentiation). (A) Proliferative cultures were incubated with 5 µM BrdU for 22 hours before fixation. (A-D) The proportions of TuJ1-positive, GFAP-positive and O4-positive cells co-labelled for GFP were calculated. The data obtained in PTEN-GFP infected cultures was expressed as the percentage of the data obtained in GFP-infected cultures, which were considered to be 100% (dotted line). The results are expressed as the average ± s.e.m. of data of 4-8 cultures from 2-5 experiments. Statistical analysis comparing values obtained with insulin (+INS) and without insulin (–INS) was performed using Student's t-test. PTEN overexpression significantly reduced the proportions of TuJ1-positive and GFAP-positive cells in the absence of insulin (*P<0.05) whereas the proportion of O4-positive cells showed a tendency to increase. (E) The proportions of TUNEL-positive cells were not affected by PTEN overexpression under any of the growth conditions tested.