Visualization of aurora B with H3S10ph in living cells and the effects of kinase and phosphatase inhibitors. (A) HeLa cells loaded with FabAuB-488 and Fab311-555. Maximum projections of three z-stack images at 1.5-µm intervals are shown. Arrows and closed arrowheads indicate FabAuB concentrations in heterochromatin, the midzone, and the midbody. Open arrowheads indicate Fab311 foci before the accumulation on condensed chromosomes (asterisks). See Video 8. (B) A fibroblast loaded with FabAuB-488 (green) and Fab311-555 (magenta). Deconvolved and maximum-projected images of five z-stack images at 0.75-µm intervals taken every 2 min are shown. Full time series images of the boxed areas are shown. (top) White arrowheads indicate FabAuB and Fab311 foci. (bottom) The positions of Fab311 foci with the highest intensities are indicated (arrowheads). See Video 9. (C) BrdU incorporation in cells exhibiting aurora B and H3S10ph. Cells were incubated in BrdU for 10 min to 5 h, fixed, and immunolabeled for aurora B, H3S10ph, and BrdU. (left) When treated with BrdU for 10 min, aurora B–positive cells often exhibit BrdU signals, which are unassociated (arrows; HeLa) or associated (arrowheads; fibroblasts) with H3S10ph. (right) BrdU-positive cells were counted among those positive for aurora B, H3S10ph, or condensed chromosomes (n > 20); cells in early mitotic phase (i.e., prophase to prometaphase; M) were counted in HeLa because of their prolonged mitosis (> 1 h vs. ∼30 min in fibroblasts). The means ± SD from three independent experiments are shown. (D) A fibroblast treated with ZM. (E) A fibroblast treated with TMC followed by ZM. (F) A HeLa cell treated with TMC. Bars, 5 µm.