Figure 4.

Rsf-1 depletion impairs deposition of CENP-A to the centromeric core chromatin. (A) Immunoblot of Rsf-1 and CENP-A in CENP-A– (a) and Rsf-1–depleted (b–d) cells. Analysis was performed in whole-cell lysate (a and b), nuclei after low salt (c), and high salt (0.6 M NaCl) wash (d). “Core chromatin” represents the high salt insoluble nuclear fraction and “nuclear extract” represents the high salt soluble nuclear fraction, prepared as schematically depicted in e. Depletion was induced by two rounds of specific siRNA transfection at d 0 and d 2 before sample preparation at d 5 for Rsf-1, and d 4 for CENP-A. The intensity of each CENP-A band of mock and siRNA-treated sample was quantified, and the intensity relative to mock sample is shown at the bottom of each lane. Histone H4 was visualized on a duplicate gel stained with Coomassie Brilliant blue. Rsf-1 = ∼250 kD; SNF2h = ∼135 kD; CENP-A = ∼17 kD. (B) The effect of high salt wash on centromere stability of CENP-A immunofluorescent signals, in siCENP-A– and siRsf-1–depleted cells. Mock-, CENP-A–, and Rsf-1–depleted cells were fixed and washed in low salt (0.15 M NaCl; top) or high salt (0.5 M NaCl; bottom). Depletion of each protein was induced by two rounds of siRNA transfection as in A. Nuclei were visualized by DAPI staining. Images showing CENP-A (green) and CENP-C (red) were merged. (C) Quantification of the CENP-A fluorescent signals on photographed cells prepared in B. The intensity of the fluorescent signal was measured for each of 300–1,000 cells per depletion type, and the mean intensity value per cell was normalized with mock sample in each washing condition. Error bars represent ± SEM.