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Fig. 7. (A) Freshly plated CGC cultures prepared from P2 cerebella were stimulated with L-NAME (1 mM), ODQ (5 µM) or KT5823 (500 nM), for 20 hours. BrdU (10 µM) was added for the last 6 hours, after which time cells were processed for BrdU incorporation by an ELISA method. Values are the mean ± s.e.m. of three experiments. *P<0.05, compared to control (Bonferroni's test after ANOVA). (B,C) Colour merged images of double immunofluorescence for III beta-tubulin (green) and BrdU (red) of control (B) and L-NAME-treated (C) CGC cultures. Plated CGC cultures were stimulated with L-NAME (1 mM) for 20 hours. BrdU (10 µM) was added for the last 6 hours, after which time cells were processed for immunofluorescence. Arrowheads point to III beta-tubulin-BrdU labelled cells. Bar, 30 µm. (D) Percentage of BrdU-labelled cells positive for the early neuronal marker, III beta-tubulin, or the glial marker, GFAP in the same cultures as A-C. Counting was done in random fields of control and L-NAME-treated cultures from two independent experiments. *P<0.01, compared to control (Bonferroni's test after ANOVA). (E) BrdU incorporation during the last 6 hours of culture was measured in CGC by ELISA method after 20 hours in culture in the presence or absence of L-NAME (1 mM) and/or Shh (3 µg/ml). Data are expressed as the mean ± s.e.m. of three experiments. *P<0.05, **P<0.01, compared to control (Bonferroni's test after ANOVA). (F) Expression of Myc in CGC cultures treated with L-NAME (1 mM) and/or Shh (3 µg/ml) for 12 hours. Total RNA was extracted from CGC cultures and equal amounts were used for real-time PCR reaction performed with specific primers for Myc, as described in the Materials and Methods section. The results, expressed as percentage of control, are the mean ± s.e.m. of two different experiments performed in triplicate. *P<0.05, **P<0.01 (Bonferroni's test after ANOVA).
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