Sds22 depletion affects aurora B activity. (A) Experimental protocol. (B) Monopolar, prometaphase, and metaphase data from monastrol recovery. Examples of cells at the three points of recovery counted in C. Overlay shows DAPI (blue), ACA (green), and microtubules (red). (C) Effect of Sds22 depletion on the recovery from monastrol arrest. 48 h after transfection of HeLa cells with control or Sds22-specific RNAi, duplexes were treated for 4 h with monastrol and released into fresh medium or medium containing 0.3 or 1 µM ZM. After 1 h, cells were fixed, stained (A), and mitotic cells were scored for their progression through mitosis. Total number of cells: 0 µM control, 347; 0 µM Sds22, 1,056; 0.3 µM control, 1,336; 0.3 µM Sds22, 1,230; 1.0 µM control, 1,261; 1.0 µM Sds22, 1,360. (D) Aurora B T232 phosphorylation depends on Sds22. HeLa cells were fixed and immunostained with anti–phospho-T232 antibody 48 h after transfection with control (con) or Sds22-specific duplexes. Overlay shows tubulin (green), anti–phospho-T232 aurora B (red), and ACA (blue). The spindle pole staining is not sensitive to inhibition with 1 µM ZM and is likely spurious and the result of cross-reaction with phospho–aurora A (Fuller et al., 2008). (E and F) Effect of Sds22 depletion on total aurora B and T232 aurora B phosphorylation in metaphase. Quantification of metaphase cells fixed and stained with aurora B (E) and anti-phospho–aurora B T232 (F) antibodies in fixed HeLa cells 48 h after transfection with control or Sds22-specific duplexes (see Materials and methods). Intensities were normalized relative to the general kinetochore marker ACA. Box plots show mean (middle line), top and bottom quartiles of the data as top and bottom of the box, and whiskers as the extent of 90% of the data. P-values were calculated using a Kolmogorov-Smirnov test. (E) Total number of cells: control RNAi, 30; Sds22 RNAi, 34. (F) Total number of cells: control RNAi, 24; Sds22 RNAi, 24. Error bars show SEM from three independent experiments. Bars, 10 µm.