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Fig. 7. Effects of blocking MAPK on neural stem cell maintenance. (A)
Phosphorylated MAPK expression and DAPI labelling of a sectioned neurosphere
grown from P0 rat brain. Note the peripheral location of the P-MAPK labelling.
Scale bar: 40 µm. (B-G) A decrease in neurosphere formation is seen in
serial dilution assays in the presence of blockers for MAPK (PD98059). B shows
the entire dilution series in a single experiment for cells grown in EGF
plotted on a log-log scale, whereas E shows the same dilution series with
linear scales and the regression lines from the data. C and F show the same
data for cells grown in FGF. Note that, as expected, the EGF receptor
inhibitor AG1478 also inhibits neurosphere formation at all dilutions in EGF.
Blockers of MAPK38 (SB203580) and PI3 kinase (wortmannin and LY294002) do not
show any effect on neurosphere formation. PD, PD98059; AG, AG1478; WM,
wortmanin; Ly, Ly294002; SB, SB203580. D and G show the effect of the MEK
inhibitor U0126 on cells grown in EGF, as compared with the control U0124 or
untreated cells. Note that inhibition of sphere formation is also seen with
this inhibitor. The efficacy of the inhibitors is shown in the western blots
(H). Lanes 1-6 and 8-13 show cells grown in EGF and FGF, respectively, and
exposed to LY294002 (lanes 1, 8), wortmannin (lanes 2, 9), U0124 (lanes 3,
10), U0126 (lanes 4, 11) or PD98059 (lanes 5, 12), or grown without inhibitors
(lanes 6, 13). Lanes 7 and 14 show cells starved of growth factors prior to
analysis. Note that growth factor deprivation reduces phosphorylation of both
MAPK and Akt (compare lanes 6, 7 and 13, 14). PD98059 and U0126 reduce only
MAPK phosphorylation, whereas wortmannin and LY294002 reduce only Akt
phosphorylation.
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