Depletion of Sds22 causes defects in mitotic progression. (A) Depletion of Sds22 by siRNAi. 70 µg extracts of logarithmically growing HeLa cells 48 h after transfection with Sds22-specific and control siRNAi were probed with Sds22-specific polyclonal antibody by Western blotting and reprobed with anti-tubulin as a loading control. (B) Cell cycle profile of HeLa cells after Sds22 RNAi. Subpopulation of HeLa in specific cell cycle stages as determined by DNA content analysis (FACS of propidium iodide–stained cells). Cells were transfected with control (blue) or Sds22-specific (red) siRNAi and analyzed at the time points indicated (hours). Results are from four separate experiments. (C) Timing of mitosis in Sds22-depleted and control cells. HeLa (Kyoto) cells stably expressing EGFP–CENP-A were imaged at 5- or 10-min intervals starting 48 h after transfection with Sds22-specific and control RNAi. Time elapsed from NEB to the onset of anaphase was plotted against cumulative percentage of cells having completed mitosis. Data were generated from three independent experiments. Data from two experiments recorded at 5-min intervals were binned to 10 min and combined with one experiment recorded at 10-min intervals. Total number of cells: control, 314; Sds22 RNAi, 293. (D) Mitotic defects in Sds22-depleted cells. Still images of C are shown with time after NEB indicated (hours:minutes). (i) Control cell. (ii and iii) Sds22-depleted cell. Defects are indicated by arrowheads. (iv) Mean percentage of mitotic cells dividing with unaligned chromosomes in C. (v) Mean percentage of mitotic cells dividing with lagging kinetochores in the midzone in C. Error bars indicate SEM. Bars, 10 µm.