Fig. 1.

RSF1 is necessary for restricting H2A-pT120 and Sgo1 to centromeres. a HeLa cells were transfected with siRNAs, and floating mitotic cells were obtained after nocodazole treatment for 4 h and subjected to metaphase chromosome spread stained with Giemsa. Quantification of sister chromatid separation in HeLa cells after depletion of RSF1 or SNF2H. Data represent mean ± SEM; **p < 0.01 vs. control siRNA by Student’s t-test. b HeLa cells were transfected with siRNAs, and RSF1-depleted cells were subjected to chromosome spread immunostaining. Images were obtained from representative mitotic cells: Sgo1 (green), ACA (red), and DAPI (blue). The percentages of cells exhibiting the arm or centromeric localizations of Sgo1 proteins are shown. Scale bar, 5 μm. c RSF1 knockout mitotic cells were analyzed by immunofluorescence staining: H2A-pT120 (green), ACA (red), and DAPI (blue). Percentage of cells exhibiting the arm or centromeric expression level of H2A-pT120 are shown. Scale bar, 5 μm. d Localization of Bub1 on mitotic chromosomes in RSF1 WT or knockout (KO) cells. Nocodazole-treated, mitotic RSF1 WT or knockout (KO) HeLa cells were stained with DAPI and the Bub1 antibodies. The graph represents relative intensity of Bub1 against ACA at kinetochores. At least 100 kinetochores of prometaphase cells were analyzed in three independent experiments. Scale bar, 5 μm. e Metaphase chromosome spreads were stained with anti-Bub1-pS969 and anti-ACA in RSF1 WT or KO cells. Cells were stained with DAPI and the Bub1-pS969-specific antibodies. The graph represents relative intensity of Bub1-pS969 against ACA at kinetochores. At least 100 kinetochores of prometaphase cells were analyzed in three independent experiments. Scale bar, 5 μm