Fig. 5. Antagonism of Nmyc (N-myc in figure) activity reduces Shh-induced proliferation. (A) Mxi inhibits proliferation in Shh-treated cultures, as measured by flow cytometry. Thirty-six hours after infection with Mxi-SR-carrying retrovirus or controls and Shh-treatment, cycle phase distribution of cells was analyzed by flow cytometry. Percentages of cells in S phase were normalized to levels in Shh-treated, uninfected/GFP infected samples. The graph depicts average normalization from n=6 samples per treatment, per timepoint. Error bars indicate standard error of the mean. Northern blot analysis confirmed strong expression of Mxi (upper panel) in CGNPs infected as indicated. Endogenous Mxi expression can also be observed. (B) The NmycMB2 mutant inhibits DNA synthesis in Shh-treated cultures, as measured by BrdU immunolabling. (Top) Immunostaining to visualize BrdU incorporation was used to measure proliferation in CGNPs infected with GFP or NmycMB2+GFP retroviruses. Representative fields of BrdU immunostaining and GFP immunostaining in GFP-infected and NmycMB2+GFP-infected CGNPs are shown. (Bottom) Graph shows levels of BrdU incorporation in untreated, GFP-infected or Shh-treated NmycMB2+GFP-infected cells, normalized to levels in Shh-treated GFP-infected CGNP cultures. Western blot analysis (right) shows expression levels of Nmyc and NmycMB2 protein levels. ^*P<0.01, in comparison with Shh-treated control CGNPs.

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