FIG. 5.
Haploid insufficiency of NS in MEFs leads to reduced proliferative potential. (A) NS endogenous protein is detected by Western blotting analysis in total extracts from E10.5 embryos (top panel) or in MEFs at various passages (lower panel). A significant decrease in NS protein expression is detected in cells prepared from NS+/− heterozygous embryos, and NS expression decreases at late passages. γ-Tubulin (γ-tub) serves as a loading control here. (B) The NS protein (green) is mainly localized in the MEF nucleoli, as assessed by indirect immunofluorescence. Nuclei are visualized using DAPI staining. (C) Growth curves for NS+/− and NS−/− MEF cultures at passage 4. Cells (1 × 105) were plated into six-well plates. Cultures were harvested at daily intervals, and the total number of cells was determined and normalized to the number of cells at day 1 (20 h after plating). The numbers refer to the mean values of four independently derived MEF cultures of each genotype. (D) Proliferation of NS+/− and NS−/− MEF cultures on a revisited 3T3 schedule. The accumulated number of population doublings is shown on a linear scale on the y axis.