Figure 3

FACS analysis of cell cycle fractions in propidium iodide stained cultured E14.5 PMEFs and disaggregated E11.5 fetuses. A-C) Relative DNA content of passage 3 PMEFs, estimated for 100,000 cells per sample. Cells were allocated to bins representing the G1- (A), S- (B) and G2- (C) phases of the cell cycle. Cell populations that were <2n and >4n were excluded from the analysis. D) Table summarising results of statistical analysis of data in A-C. All values represent means ± SEM, one way ANOVA with Tukey’s post-hoc analysis. WT n = 6, Dlk1 ^+/p n = 5, Grb10 ^m/+ n = 7, Grb10 ^m/+/Dlk1 ^+/p n = 6; E-G) Cells derived directly from wild type, Dlk1 ^+/p, Grb10 ^m/+ and Grb10 ^m/+/Dlk1 ^+/p E11.5 fetuses were used to estimate relative DNA content for 100,000 cells per sample. Cells were allocated to bins representing the G1- (E), S- (F) and G2- (G) phases of the cell cycle. H) Table summarising results of statistical analysis of data in E-G. All values represent means ± SEM, one way ANOVA with Tukey’s post-hoc analysis. WT n = 7, Dlk1 ^+/p n = 8, Grb10 ^m/+ n = 6, Grb10 ^m/+/Dlk1 ^+/p n = 5. For both PMEFs (A-D) and E11.5 fetal cells (E-H) significantly lower percentages of both Grb10 ^m/+ and Grb10 ^m/+/Dlk1 ^+/p cells were found in S-phase of the cell cycle in comparison to either wild type (PMEFs) or Dlk1 ^+/p (E11.5 embryo) cells. Conversely, significantly higher percentages of both Grb10 ^m/+ and Grb10 ^m/+/Dlk1 ^+/p cells were found in G2-phase of the cell cycle in comparison to wild type and Dlk1 ^+/p- E11.5 embryo cells. Similar trends were seen for PMEFs but only the comparisons of Grb10 ^m/+ and Grb10 ^m/+/Dlk1 ^+/p with wild type reached significance. No significant differences were observed for numbers of cells in G1 for comparisons between any of the four genotypes; * P <0.05. ANOVA, analysis of variance; E, embryonic day; FACS, fluorescence activated cell sorting; PMEF, primary mouse embryonic fibroblasts; SEM, standard error of the mean; WT, wild type.