Presence of a spherical mitotic spindle in endomitotic MKs. Cultures were performed as described in Fig 1 in the absence of nocodazole. Cells were centrifuged on a slide, fixed with a microtubule stabilizing buffer containing 3% formaldehyde, and then permeabilized with 0.2% saponin. Cells were labeled with an anti-tubulin antibody (a, b, c, d, e, f, g, h, and k), an anti-vWF antibody (a), propidium iodide (c), an anti-kinetochore antibody (i and k), and the 3F3 MoAb (j). Cells were then examined by confocal microscopy (original magnification × 1,000 for [a] through [j] and × 1,500 for [k]). (a) Superimposition of a double staining with the anti-tubulin MoAb (FITC) and the anti-vWF (TRITC). (b) Anti-tubulin staining shown alone. Note that the complex mitotic spindle has a spherical conformation. (C) Superimposition of a double staining with the anti-tubulin MoAb (FITC) and propidium iodide. (d) Anti-tubulin staining shown alone. (e, f, g, and h) A mitotic spindle shown under different angles. Spindle microtubules radiate from each aster to form a spherical conformation. (i and j) Double staining with the anti-kinetochore antibody (TRITC) and 3F3 MoAb (FITC). Kinetochores are distibuted all around and the 3F3 labeling only colocalized with the asters, as previously demonstrated during anaphase of a mitotic cycle.40 (k) Double staining with the anti-kinetochore antibody (TRITC) and the anti-tubulin MoAb (FITC). The kinetochores are clustered around each aster, suggesting that chromosome segregation has occured.