Figure 4. Centrosome and mitotic spindle abnormalities are elevated in Cenpj-deficient cells.

A. Images show examples of Cenpj staining in centrosomes of Cenpj ^+/+ and Cenpj^tm/tm mouse embryonic fibroblasts (MEFs). Cells were stained with antibodies against Cenpj (green in merge) and the centrosomal protein γ-tubulin (red in merge). Framed areas are shown at higher magnification. B. Graph shows mitotic spindle phenotypes in MEFs derived from Cenpj ^+/+, Cenpj ^+/tm and two independent Cenpj^tm/tm embryos (littermates, +/+ MEFs passage 4, +/tm and tm/tm MEFs passage 3): tm/tm (1) and tm/tm (2). Number of mitotic cells scored are shown for each genotype. Examples for monopolar and multipolar spindle are shown. Note cell on bottom panels forming a bipolar spindle by clustering supernumerary centrosomes. Cells were stained with antibodies against α-tubulin (green in merge) and the centrosomal protein, Cdk5RAP2 (red in merge). C. Graph shows centriole numbers in mitotic MEFs of indicated genotypes (littermates, +/+ MEFs passage 4, +/tm and tm/tm MEFs passage 3). Cells were arrested in mitosis with monastrol that caused monopolar spindle formation and facilitated visualization of centrioles. Note that mitotic cells should normally contain a total of 4 centrioles, but even in wild-type cells we occasionally detect 3 centrioles probably due to insufficient spatial resolution, so 3 or 4 centrioles were considered a single class. Data were collected from two independent experiments; bars show mean ±SD, number of mitotic cells scored are shown for each genotype. Images below depict examples for cells with different centriole numbers (top cell with 4 centrioles is normal, all other cells have too few or too many centrioles). Cells were stained with antibodies against the microtubule-binding protein Tpx2 (green in merge) and the centriolar protein, centrin-3 (red in merge). Framed areas are shown at higher magnification. Scale bars = 5 µm.