FAM29A is a spindle protein preferentially associated with kinetochore MTs. (A) HeLa S3 cells were synchronized at the G1/S boundary by a double-thymidine arrest and harvested at the indicated time after release. Cell cycle profile was analyzed by FACS with anti-MPM2 antibody staining and propidium iodide staining. Protein levels were determined by Western blotting with Hsp70 as a loading control. (B–E) Maximum projections from deconvolved z stacks of representative HeLa cells (B, D, and E) or a HeLa cells transiently expressing GFP-FAM29A (C). Cells were stained for FAM29A (B, D, and E)/GFP (C) (green), β-tubulin (B–D)/centrin (B)/Hec1 (E) (red), and DNA (blue). In D, the cell was incubated with Monastrol (4 μM) for 4 h and then stained. The inset in E shows a single focal plane of the boxed region. (F) HeLa cells were cultured at 37°C and then incubated at 4°C for the indicated time. Cells were stained for FAM29A, β-tubulin, and DNA. MT and FAM29A fluorescence intensity in the mitotic spindle of metaphase cells (n = 10 cells for each time point) were quantified and normalized to their respective intensity at the 0-min time point. Error bars, SEM. Bars, 5 μm.