FAM29A controls the maturation of kinetochore MT fibers. (A) HeLa cells transfected with siRNAs were incubated at 4°C for 10 min, fixed, and stained for CREST (green), β-tubulin (red), and DNA (blue). Shown are maximum projections from deconvolved z stacks of representative cells. Insets show single focal planes of the boxed regions. Total intensity of spindle MTs as well as the intensity of k-MTs (the MT signal from a defined area immediately next to the CREST signal) were quantified and plotted (n = 10 metaphase cells for each quantification). *, P < 0.05; **, P < 5.6 × 10−5 (two-tailed t test relative to siControl metaphase cells). AU, arbitrary units. (B–D) HeLa cells transfected with siRNAs were incubated at 37°C or at 4°C for 10 min, fixed, and stained for CREST (B)/Mad2 (C and D) (green), β-tubulin (B)/Hec1 (C and D) (red), and DNA (blue). Shown in B and C are maximum projections from deconvolved z stacks of representative cells. Insets show single focal planes of the boxed regions. Fluorescence intensities of kinetochore Mad2 in prometaphase or metaphase cells under different conditions were quantified and plotted in D (n = 120 kinetochores). For metaphase cells in D, only kinetochores on chromosomes aligned at the metaphase plate were quantified. *, P < 1.06 × 10−4; **, P < 1.14 × 10−7 (two-tailed t test relative to siControl metaphase cells). Error bars, SEM. Bars, 5 μm.
