Lethality to human cancer cells through massive chromosome loss by inhibition of the mitotic checkpoint

Kops et al. 10.1073/pnas.0401142101.

Supporting Information

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Supporting Figure 6
Supporting Movie 1
Supporting Movie 2
Supporting Movie 3
Supporting Movie 4
Supporting Movie 5
Supporting Movie 6
Supporting Figure 7
Supporting Figure 8

Supporting Figure 6

Fig. 6. Small interfering RNA (siRNA)-mediated reduction of BubR1 and Mad2 protein. (A and B) HeLa or T98G cells transfected with mock or BubR1 siRNA (A) or Mad2 siRNA (B) plasmid for 24 or 48 hr were isolated by magnetic-activated cell sorting (MACS) and analyzed for BubR1 or Mad2 protein, respectively, by serial dilutions of whole cell lysates. The label 100% lysate indicates 20 m g (A) or 80 m g (B) of protein lysate. The immunoblots of T98G lysates were from whole-cell lysates of the entire cell population. (C) HeLa cells transfected with mock or BubR1 siRNA (Upper) or Mad2 siRNA (Lower) plasmids were isolated by MACS and replated for 24 hr (Upper), or cotransfected with pH2B-EYFP (Lower). Cells were fixed, extracted, and stained for BubR1, CENP-E, and DNA (DAPI) (Upper), or for Mad2 and centromeres (ACA) (Lower). (Insets) Kinetochores.

Supporting Figure 7

Fig. 7. Reduction of Mad2 or BubR1 protein by additional siRNA constructs abolishes checkpoint signaling. Shown are stills of live cell microscopy of HeLa cells transfected with pH2B-EYFP and BubR1 siRNA no. 2 (sequence: 5'-GAGACTGCACAACAGCCAG-3') or Mad2 siRNA no. 2 (sequence: 5'-TCCGTTCAGTGATCAGACA-3'). Twenty-four hours posttransfection, images of H2B-EYFP-positive cells were taken at 2-min intervals. Arrows indicate unaligned chromosomes during anaphase.

Supporting Figure 8

Fig. 8. Chromosome loss during mitotic division in absence of BubR1 or Mad2. Shown are stills of time-lapse movies of HeLa cells transfected with BubR1 or Mad2 siRNA plasmid in combination with pH2B-EYFP. Forty-eight hours posttransfection, fluorescent and brightfield images of H2B-EYFP-positive cells were taken at 2-min intervals. Arrows indicate chromosomes lost for both daughter cells, and black dots in brightfield images correspond to position of lost chromosomes.

Supporting Movie 1
Supporting Movie 2
Supporting Movie 3

Movies 1-3. Live-cell microscopy of HeLa cells transfected with either mock (Movie 1), BubR1 siRNA (Movie 2), or Mad2 siRNA (Movie 3) plasmid in combination with pH2B-EYFP. Forty-eight hours posttransfection, colcemid was added for 5 min, and images of H2B-EYFP-positive cells were taken at 2-min intervals.

Supporting Movie 4
Supporting Movie 5
Supporting Movie 6

Movies 4-6. Live-cell microscopy of HeLa cells transfected with either mock (Movie 4), BubR1 siRNA (Movie 5), or Mad2 siRNA (Movie 6) plasmid in combination with pH2B-EYFP. Forty-eight hours posttransfection, images of H2B-EYFP-positive cells were taken at 2-min intervals.