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Fig. 2. Hippocampal cultures express markers of cell cycle inhibition and neuronal
differentiation after incubation in CMCerebellum. (A-D) Western
blots and/or immunofluorescence staining show that addition of
CMCerebellum (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 CMCerebellum. (E)
CMCerebellum 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
CMCerebellum were exposed to BrdU for 8 hours, and washed and
maintained in CMCerebellum 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 CMCerebellum. Numerical data refer to
mean±s.d. (n=4-6) *P<0.05, **P<0.01,
***P<0.001 (versus appropriate controls).
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