Fig. 2. Hippocampal cultures express markers of cell cycle inhibition and neuronal differentiation after incubation in CM_Cerebellum. (A-D) Western blots and/or immunofluorescence staining show that addition of CM_Cerebellum (10 µl/ml) to hippocampal cultures induces expression of the cyclin-dependent kinase inhibitors p21 and p27 (A) and of the mature neuron marker MAP2A/MAP2B (B,C) with concomitant decreases in the expression of the neuroblast marker, doublecortin (D). Insets show examples of immunoblots for p21 (A) and p27 (A) and MAP2A/MAP2B (B), as well as for actin (which served as an internal reference). Results shown in A-D were obtained after 24 hours treatment with CM_Cerebellum. (E) CM_Cerebellum treatment influences proliferation and differentiation of neurons as shown by the significantly increased number of MAP2A/MAP2B-positive/BrdU-positive double-stained cells relative to the total number of BrdU-positive cell population. For this analysis, cells growing in CM_Cerebellum were exposed to BrdU for 8 hours, and washed and maintained in CM_Cerebellum for 72 hours before fixing and processing for MAP2A/MAP2B and BrdU double immunocytochemistry. There was no statistical difference on the incidence of apoptosis in cultures grown in either control medium or CM_Cerebellum. Numerical data refer to mean±s.d. (n=4-6) *P<0.05, **P<0.01, ***P<0.001 (versus appropriate controls).