Prometaphase centrosome separation and centrosome-independent spindle pole focusing require NuMA. (A) Primary MEFs were arrested in mitosis by treatment with STLC and processed for immunofluorescence. Green, α-tubulin; blue, DNA; red, γ-tubulin. (B) Frequencies of monopolar spindles as shown in A. Two independent cell lines per genotype were used, and >200 mitoses per genotype were counted. (C) Anaphase in NuMA^+/Δ22 control and NuMA^Δ22/Δ22 cells 1 h after washout of STLC. In merged images, DNA is shown in purple and γ-tubulin in green. Arrows indicate centrosomes. (D) Stills from videos of NuMA^+/Δ22 (Video 3, available) and NuMA^Δ22/Δ22 (Video 4) primary embryo fibroblasts transduced with retrovirus encoding tubulin-YFP. Cells were arrested with STLC for 3–4 h and filmed after washout of the drug. Scoring for centrosome separation was performed blinded to genotype. Each time point shows a maximum intensity projection of 12 confocal fluorescence z sections acquired in 1-µm intervals. Error bars indicate SEM.