Depletion of haspin causes a failure of chromosome congression. (A) U2OS cells transfected with haspin siRNA (Ambion ID #1093) or control siRNA (Ambion #4613) were fixed and stained with DRAQ5 to visualize DNA and with human centromeric autoantibodies followed by antihuman IgG-Cy3 (red), mouse anti-α-tubulin mAb followed by anti-mouse IgG-Alexa488 (green), and rabbit anti-phospho-H3 (Thr-3) followed by anti-rabbit IgG-Alexa488 (green) or anti-rabbit IgG-Cy3 (red) as indicated. (B) U2OS cells were transfected with haspin siRNA A (Ambion ID #1093), haspin siRNA B (Dharmacon SMARTpool), control siRNA A (Ambion #4613), or control siRNA B (Dharmacon SMARTpool), or without siRNA. After 48 h, the cells were fixed and stained with propidium iodide, and ∼3000 cells were counted on each of three coverslips for each condition and classified as interphase, prophase, prometaphase, metaphase, anaphase/telophase, or partial metaphase (a metaphase plate and three or more unaligned chromosomes; a subset of prometaphase) by the distinctive pattern of DNA staining. The percent of mitotic cells in each phase is shown. The increase in prometaphase and partial metaphase cells and the decrease in anaphase/telophase cells have p < 0.0001 by two-tailed Student's t-test when comparing haspin siRNA A and B with control siRNA A, B, and no siRNA combined. (C) Haspin siRNA transfected U2OS cells in prometaphase and metaphase from B stained with rabbit anti-phospho-H3 (Thr-3) followed by anti-rabbit IgG-Alexa488 were further classified as containing low or high levels of phosphorylated H3 Thr 3 (pT3). The percentage of these cells that were in prometaphase, metaphase, or partial metaphase is shown.