Loss of cohesin from the mitotic centromere by HP1-αγ RNAi. (A) Giemsa-stained chromosomes from HeLa cells transfected with control (Cont), HP1-α, HP1-γ, or combined HP1-α + HP1-γ (HP1-αγ) siRNA (48 h). (B) PCS frequencies. (C) Frequency of hRad21-myc8–positive cells among HH2B-EGFP–positive cells transfected with control or HP1-αγ siRNA. (D) Control or HP1-αγ siRNA–transfected HeLa cells were cotransfected with hRad21-myc8 and histone H2B-EGFP (HH2B-EGFP). Chromosome spreads were stained for c-myc (red) and EGFP (green). Similar results were obtained in three independent experiments. (E) Giemsa-stained chromosome spreads from cells transfected with no RNA (control) or with hSgo1 siRNA (48 h). ΔVpr cells + DOX (top), MIT-23 cells + DOX (top middle), HeLa cells and control RNAi (bottom middle), and HeLa cells and HP1-αγ RNAi (bottom) are shown. (F) PCS frequencies in cells transfected with hSgo1 or control siRNA with or without Vpr expression. (G) PCS frequency in HeLa and HT1080 cells transfected with hSgo1 or control siRNA with or without HP1-αγ RNAi. (H and I) Cells were transfected with control or hSgo1 siRNA with or without HP1-αγ RNAi followed by hRad21-myc8 transfection (48 h). (H) Chromosome spreads stained for c-myc (red) and EGFP (green). (I) Frequency of myc-negative cells among HH2B-EGFP–positive cells. (H and I) Similar results were obtained in two independent experiments, and representative data are shown. (B, C, F, and G) Values represent the mean ± SD of three independent experiments. Bars: (A and E) 5 µm; (D and H) 10 µm.