Figure 5: Lack of Diaph3 disables the SAC.

(a–d) Immunostaining for BubR1 (green) and pHH3 (red) of cortical sections from WT (a) and Diaph3 ko (c) E10.5 embryos. (b,d) Enlargements of the boxed areas in a,c, respectively. BubR1 (a hallmark of SAC activation) accumulated more in normal than in Diaph3-deficient cells (quantification in e; n=1,473 control cells from 3 animals and 1,144 ko cells from 3 animals; P<0.0001, z-test). (f) The density of mitotic cells in the cortex of E10.5 embryos was lower in the ko than in WT (n=48 WT and 49 ko of 100 μm wide cortical stripes from 3 animals each genotype; P<0.0001; Student’s t-test). (g) Quantification of postmetaphasic cells in the population of mitotic cells. The percentage of mitotic cells that underwent the metaphase–anaphase transition was higher in the ko than in control littermates (n=786 control cells from 6 animals and 889 ko cells from 5 animals; P<0.0001, z-test). (h,i) Reduction of BubR1 protein levels in the mutant telencephalon detected by western blotting and quantified relatively to tubulin. The same membranes were blotted for Diaph3 to confirm the absence of the protein (h). The level of BubR1 decreased by half in the ko (i; n=15 embryos in 4 pools for each genotype). (j) Western blotting (WB) detects BubR1 on IP with anti-Diaph3 antibodies from TG cortical lysates. Diaph3 was used as a positive control for the IP (Input). No signal was found in the eluted fraction (flow-through, FT) in presence of Diaph3 antibodies. Detection of the protein in the lysate (Input) required overexposure of the film. Scale bars, 50 μm (a,c) and 5 μm (b,d). Error bars represent s.e.m.