Figure 3.

siRNA-mediated depletion of Rsf-1 and SNF2h of RSF delays cell cycle progression and causes kinetochore misalignment. (A) Immunostaining of HeLa cells with anti-Rsf-1 (top panels) and anti-SNF2h antibodies (bottom panels) at 48 h after mock and specific siRNA transfection. (B) Western blot analysis of Rsf-1 and SNF2h levels in cells siRNA-depleted of Rsf-1 (right) or SNF2h (left) at 0, 24, 48, and 72 h post-transfection. WB, Western blot; CBB, Coomassie Brilliant blue staining of a gel-duplicate. Rsf-1 = ∼250 kD; SNF2h = ∼135 kD. (C) Results of microscopic observation of cell stage distribution in G2 (blue), prometaphase (green), metaphase (red), and anaphase/telophase (yellow) at 2–6 d after siRNA-transfection of Rsf-1, SNF2h, and co-depletion (RSF) (n = 200–400 cells/depletion type/day post-transfection). Discrimination of each cell stage was based on the shape and condensation degree of chromosomes, distribution pattern of centromeres, and/or presence or absence of mitotic microtubules. Differences between Pm and M are shown in E as an example. (D) Prometaphase to metaphase cell ratio at 4 d post-transfection for each depletion type in C, and siCENP-A–transfected cells (see also Fig. S3). (E) Co-immunofluorescent staining of HeLa prometaphase and metaphase cells in control and siRNA-depleted samples using anti-β-tubulin (green) and anti-CENP-C (red) antibodies. DNA is shown in blue. (F) Percentage of abnormal prometaphase and metaphase cells at 4 d post-transfection, from C (n = 1).