FIGURE 1.

And-1 depletion leads to mitotic arrest with chromosome congression defects. A, whole cell extracts from HCT116 cells transfected by two independent siRNAs (siAnd-1-1 and siAnd-1-2) were subjected to immunoblotting using the indicated antibodies. B, And-1-depleted cells showed two morphologically distinct cell populations: flat adherent cells and round mitotic cells. Phase-contrast imaging of HCT116 cells harvested 72 h post siRNA transfection is shown. C, mitotic index of And-1-depleted HCT116 cells was determined by FACS using phospho-histone H3 (Ser-10) antibody. The percent cells with P-H3 (Ser-10) is the ratio of the number of P-H3 W(Ser-10)-positive cells to total amount of cells. Data represent the means ± S.D. (error bars) from two independent experiments. D, distribution of mitotic stages in And-1-depleted HCT116 cells is shown. Counting was performed on cells stained for tubulin and DAPI. Two independent experiments were performed, and at least 300 cells were counted for each condition. Data represent the means ± S.D. E, chromosome congression defects appear after And-1 depletion. Two abnormal mitotic cell populations were observed: metaphase cells where most of the chromosomes aligned at the metaphase plate with few unaligned chromosomes or metaphase cells with severe chromosome alignment defects as indicated by randomly distributed chromosomes along the mitotic spindle. Cells treated as in A were stained for tubulin (green), And-1 (red), and DAPI (blue). Scale bar, 10 μm.