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Fig. 2. Alanine replacement of Nmyc1 T50 or S54 residues results in enhanced
proliferative effects and delayed CGNP cell cycle exit. (A) After 24 hours of
Shh treatment alone (no serum), CGNP cultures were infected with the indicated
viruses and forskolin (10 µM) was added 24 hours later. After a further 24
hour incubation period, BrdU was added (2 hour pulse) and proliferation was
assessed by BrdU immunostaining. The histogram shows levels of BrdU
incorporation normalized to Shh-treated, control CGNP cultures as previously
described (Kenney et al.,
2003; Kenney and Rowitch,
2000). Wild-type Nmyc1 is sufficient for sustained proliferation
in the presence of forskolin (fsk). Nmyc1T50A and Nmyc1S54A supported
proliferation to a greater extent than wild-type Nmyc1, despite treatment with
fsk. The western blot shows relative Nmyc1 protein levels. (B) Nmyc1T50A and
Nmyc1S54A extend the phase of CGNP proliferation relative to Shh treatment
alone. CGNP cultures were infected as indicated. Cell cycle phase distribution
was assessed by propidium iodide flow cytometry at 48 hours (left) and 96
hours (right) after infection. The average percent of cells in S
phase±s.e.m. derived from three independent experiments is shown in the
histograms above representative western blots demonstrating relative Nmyc1
protein levels. Notice that, 96 hours after infection, the proportion of cells
in S phase in Nmyc1T50A- and Nmyc1S54A-infected cultures, but not wild-type
Nmyc1-infected cultures, remained significantly higher than Shh-treated
controls. Western blots below the graphs show relative Nmyc1 protein levels.
(C) Nmyc1-, Nmyc1T50A- and Nmyc1S54A-infected cells all show upregulation of
cyclins D1 and D2 at 48 hours and 96 hours after infection. Representative
western blot autoradiographs are shown. ß-Tubulin immunoreactivity
indicates equivalent loading of the lanes.
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