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Fig. 9. The effect of Wnt3a on cell cycle progression and the effect of U0126 on NIH3T3 cells. (A) NIH3T3 cells were grown in DMEM and synchronized with double thymidine blocking (Park et al., 2002). Cells were either not treated or treated with 150 ng/ml recombinant Wnt3a for 24 hours before harvest for FACS analysis. Where required, 20 µM U0126 was added 1 hour before treatment with Wnt3a. Error bars indicate the standard deviations of three independent experiments. (B) The effect of U0126 on Wnt3a-induced BrdU incorporation in NIH3T3 cells. NIH3T3 cells were grown in DMEM and either not treated or treated for 24 hours with 150 ng/ml of recombinant Wnt3a in DMEM containing 1% FBS. Where required, 20 µM U0126 was applied 1 hour before treatment with Wnt3a. Cells were labeled with 20 µM BrdU for 5 hours prior to cytochemical analysis using anti-BrdU antibody. Cell nuclei were stained with DAPI. Cells containing BrdU incorporated into the nucleus were scored as BrdU positive and the relative percentage of BrdU-positive cells was determined. Analyses were performed at least three times and 100 cells were counted in each case. Error bars indicate the standard deviations of three independent analyses.
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