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. 2006 Jan 27;7(4):425–430. doi: 10.1038/sj.embor.7400624

Figure 5.

Figure 5

Depletion of Cdc6 in S phase does not affect G2/M progression and chromosome condensation, but results in abnormal spindle formation and aberrant chromosomal congression. (A) HeLa cells grown on coverslips were synchronized and transfected with luciferase (Luc) or Cdc6 endonuclease-prepared small interfering RNA (esiRNA) at early S phase. Cells were fixed at hourly intervals and immunostained with an anti-phospho-histone-H3 antibody. The percentages of phospho-H3-positive cells were determined by microscopic evaluation of >300 cells/sample. (B) HeLa cells grown on coverslips were synchronized and transfected with esiRNA at early S phase. Cells were pulse-labelled with 10 μM 5-bromo-2′-deoxyuridine (BrdU; 1 h), fixed at hourly intervals and immunostained with anti-BrdU and anti-phospho-H3 antibodies. The percentages of BrdU/phospho-H3-positive cells at the indicated time points were scored (>250 cells) using a fluorescence microscope. (C) Cells were pulse-labelled with BrdU at 11–12 h post transfection, and were fixed and immunostained with the indicated reagents. DAPI, 4,6-diamidino-2-phenylindole.