Spindle defects in primary NuMA^Δ22/Δ22 fibroblasts. Primary fibroblasts were processed for immunofluorescence on experimental day 5, as shown in Fig. 2 A. (A) Example of metaphase in a control cell (NuMA^+/+) and two exon 22–deleted (NuMA^Δ22/Δ22) primary fibroblasts. Arrows indicate centrosomes. (B) Anaphase in wild-type and two NuMA^Δ22/Δ22 primary cells. Each image represents a maximum intensity projection of a deconvolved series of z sections spanning the entire cell in 0.2-µm intervals. Arrows indicate centrosomes. Tubulin is shown in green, and phosphorylated histone H3 is shown in purple. (C) Frequencies of spindle–centrosome dissociation and pole splaying defects seen in control and NuMA-depleted metaphase cells. Cells were scored as phenotypic if at least one centrosome was nonpolar or at least one pole displayed an obvious lack of microtubule focusing. Two independent cell lines were examined per genotype, with >50 cells counted for NuMA^+/+ and >130 cells for each of NuMA^+/Δ22 and NuMA^Δ22/Δ22 fibroblasts. (D) Spindle length in MG132-arrested primary fibroblasts measured as the linear distance between spindle poles or the approximate position of most spindle microtubule ends in defocused poles. At least 20 spindles per genotype were examined. *, P < 0.01 using Bonferroni's multiple comparison test and compared with NuMA^Δ22/Δ22; **, P < 0.001. Error bars indicate SEM.