FAM29A controls mitotic progression. (A) HeLa cells and HeLa/GFP-FAM29A cells were transfected with either control (siControl) or FAM29A-specific siRNAs (siFAM29A-A and B) and analyzed by Western blotting. (B and C) HeLa/GFP-H2B cells were transfected with siRNAs and imaged for GFP by time lapse, starting from 50 h after transfection. Cell images were captured every 3 min to monitor mitotic progression. Representative still images are shown in C and the time stamp is in h:min:s (Video 1 for siControl, and Video 2 for siFAM29A-A; Videos available). The time when a cell enters metaphase is set to 0 min. Mitotic cells were divided into different categories based on the duration of metaphase (from the initial metaphase plate formation to anaphase onset) (30 cells quantified for each transfection) (B). (D–G) A single focal plane from deconvolved z stacks of representative HeLa cells transfected with siRNAs and stained for CREST (green), Hec1 (D)/BubR1 (F) (red), and DNA (blue). Insets show the details of the boxed regions. Inter-kinetochore (Inter-KT) distances (E; n > 100 kinetochore pairs from five cells for each quantification) and kinetochore BubR1 signals (G; n = 100 kinetochores) in prometaphase and metaphase cells were quantified and plotted. In E: *, P < 4.7 × 10−24; in G: *, P < 5.0 × 10−4 (two-tailed t test relative to siControl metaphase cells). Error bars, SEM. Bars: 10 μm (C); 5 μm (D and F).
