Fig. 4. Analysis of chromosome morphology in cells depleted of condensins. (A) Metaphase plates in live HeLa cells stained with Hoechst 33342 after transfection with the indicated siRNAs. Single confocal sections of 3D-stacks at 1.6 µm slice-thickness of representative examples (n=10 for each condition) are shown. (B) Chromosome spreads from condensin-depleted cells. HeLa cells were transfected with control, CAP-D2, CAP-D3 or Smc2 siRNAs for 48 hours. Mitotic cells were collected without treatment (left and middle panels) or after 4 hours of nocodazole treatment (right panel), and incubated with either isotonic buffer (left panels) or with hypotonic buffer (middle and right panels), and fixed with Carnoy solution. Chromosome spreads made on slide-glass were stained with Giemsa. (C) Quantification of the number of spread cells whose chromosome arms had opened or remained closed after 4 hours of nocodazole treatment. 200 cells were assessed for each experiment.