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. 2011 Jun 10;19(1):132–143. doi: 10.1038/cdd.2011.62

Figure 2.

Figure 2

Abrogation of Dido3 causes chromosome segregation defects and provokes a DNA damage response. (a) Centrosome number was determined by staining for γ-tubulin and centrin (red dots) on cytospin slides of disaggregated Wt/Wt and ΔCT/ΔCT embryos at E7.5. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue background). (b) The incidence of centrosome anomalies in these embryos was quantified as the presence of more than two or less than one centrosome per cell as a percentage of total cells analyzed. Data were obtained from four mice per genotype and analyzed for statistically significant differences using the Fisher exact test. (c) Sections from Wt/Wt and ΔCT/ΔCT embryos at E7.5 were assayed for DNA damage (cells positive for phosphorylated γ-H2AX, brown dots) and apoptosis (TUNEL-positive cells, green dots). Arrowheads indicate areas of positive staining in the epiblast. Nuclei were DAPI-counterstained (blue). DAPI and TUNEL staining were performed on the same sections of a paraffin-embedded embryo, whereas γ-H2AX detection was performed in a different embryo. (d) Sections from Wt/Wt and ΔCT/ΔCT embryos at E7.5 were assayed for proliferation by monitoring BrdU incorporation 1 h post-BrdU injection into pregnant mice, followed by fluorescein isothiocyanate-anti-BrdU staining (green). Nuclei were DAPI stained (blue). Selected areas of the epiblast (indicated by numbers 1 and 3 in Wt/Wt and ΔCT/ΔCT embryos, respectively) and trophoblast (2 and 4 in Wt/Wt and ΔCT/ΔCT embryos, respectively) are shown at higher magnification. (e) p21 and p16 expression levels were assayed by RT-PCR in tissue homogenates of Wt/Wt and ΔCT/ΔCT embryos at E7.5. Data show mean±S.E.M. from three mice per genotype, each in triplicate and normalized to 18S mRNA expression. Statistical significance was calculated using Student's t-test. (f) Representative western blot analysis of Wt/Wt and ΔCT/ΔCT embryo lysates at E7.5 for the active growth-promoting form of retinoblastoma protein phosphorylated at serines 807/811 (pRb), using β-actin as loading control