FAM29A controls MT polymerization in the spindle. (A) Still frames from confocal time-lapse microscopy of HeLa/GFP–α-tubulin cells transfected with siRNAs. Cells were treated with nocodazole for 15 min, washed with PBS, and released into fresh media (t = 0 min) (Video 9 for siControl and Video 10 for siFAM29A-A; Videos available). Time, min:sec. (B–E) HeLa cells transfected with siRNAs were treated with 1 μg/ml nocodazole for 15 min, washed, and released into fresh media for the indicated time. Shown are maximum projections from deconvolved z stacks of representative cells stained for FAM29A (green), β-tubulin (B)/NEDD1 (D) (red), and DNA (blue). Fluorescence intensities of β-tubulin (C) and NEDD1 (E) were quantified and plotted (n = 10 mitotic cells). *, P < 0.003 (two-tailed t test relative to siControl cells). (F–H) Maximum projections from deconvolved z stacks of representative HeLa cells transfected with siRNAs and stained for FAM29A (green), EB1 (red), and DNA (blue) (F). The EB1 fluorescence density inside and outside the spindle (G), as well as the ratio of inside vs. outside spindle (H) were quantified and plotted (n = 10 mitotic cells for each quantification). The EB1 fluorescence density is defined as the EB1 fluorescence intensity in a fixed circular area. *, P < 0.005; **, P < 0.008; ***, P < 0.003 (two-tailed t test relative to siControl metaphase cells). Error bars, SEM. Bars: 10 μm (A); 5 μm (B, D, and F).