(A—D) HeLa cells transfected with control siRNAs or siRNAs directed against HsSAS-6, stained with antibodies against centrin-3 (red) and α-tubulin (green); DNA is shown in blue. Insets are magnified 5-fold. Scale bar, 10 μm. Cells were classified into the following categories: two pairs of centrin foci, bipolar spindle (A); two single centrin foci, bipolar spindle (B); one single centrin focus, bipolar spindle (C); one single centrin focus, monopolar spindle (D).
(E) Distribution of cells in the categories described above (n = 50 and n = 100 for 48 hr and 72 hr time points, respectively); Other: cells that could not be assigned to any of the four categories (∼5% of cells contained more than four centrin foci, for each experimental condition, and some cells contained a pair of juxtaposed centrin foci in the case of cells targeted with HsSAS-6 siRNAs). Note that monopolar spindle assembly was also observed with an independent siRNA (Leidel et al., 2005).
(F) HeLa cells expressing centrin-GFP were treated with siRNA directed against HsSAS-6 and observed 72 hr after transfection. A cell with a monopolar spindle and single centrin focus (arrow) was first imaged by DIC and fluorescence microscopy (scale bar, 10 μm), then fixed and analyzed by serial section EM (1—6: selected sections spanning the entire centriole; scale bar, 500 nm).