Figure 2. HsSAS-6 Localizes to the Proximal Part of the Procentriole.

(A and B) HeLa cells transfected with control siRNAs or siRNAs directed against HsSAS-6 and stained with antibodies against α-tubulin (green) and HsSAS-6 (red); DNA is shown in blue. Insets are magnified 5-fold. Scale bar, 10 μm.

(C) Control siRNA and HsSAS-6 siRNA-treated HeLa whole-cell lysates were analyzed by western blot using HsSAS-6 antibodies (top); the blot was reprobed with antibodies against α-tubulin as a loading control (middle). Cells treated in the same manner were analyzed by FACS (bottom). Note that there is no significant difference in cell cycle distribution between cells treated with control and HsSAS-6 siRNAs.

(D and E) HeLa cell in G2 expressing centrin-GFP and stained with antibodies against GFP (green; shown in D and E), C-Nap1 (blue; shown in D and E), and HsSAS-6 (red; shown in E). The orientation of the centrioles (large rectangles) and the procentrioles (small rectangles) is illustrated in gray. Scale bar, 500 nm. (F and G) Distances between the center of the centrin-GFP, C-Nap1, and HsSAS-6 signals (schematically represented in F) were measured for ten centriole/procentriole pairs (imaged as in E) and are reported in (G). Error bars, 95% confidence intervals. Value C is highlighted in dark gray and corresponds to the distance separating the center of the HsSAS-6 signal from the axis of the centriole (see F).

(H and I) HeLa cells processed for immunoelectron microscopy and labeled with HsSAS-6 antibodies. The centriole (viewed in cross-section), oval contours, and the procentriole (viewed laterally), rectangular contours, are shown in gray, and all the gold particles are highlighted in yellow. The structure of procentrioles and centrioles is not fully preserved due to methanol fixation. Scale bar, 200 nm.