Figure 6

The S-phase fraction of cells increases with loss of an extra 3p21 copy. Living cells of the MCHs #3/KH3 and Δ3/KH39 were incubated with BrdU in final concentration 30 μg/ml during 1 hour followed by fixation with methanol: acetic acid (3:1). FISH with biotin-labeled 3p21-specific RP6-123i13 probe and centromere-specific probe was performed to analyze the #3/KH3 and the Δ3/KH39 respectively. The probe signal was developed using Cy3-conjugated streptavidin (red), while FITC-conjugated anti BrdU antibodies (Roche Molecular Biochemicals Mannheim, Germany) identified cells in S-phase (green). Percentage of S-phase cells in subpopulations of cells with different 3p21 copy numbers in the #3/KH39 and with different chr3 copy number (counted by number of centromeres) in the Δ3/KH39 was calculated in more than 100 nuclei. The calculation was repeated three times when number of cells allowed. The average values and standard deviations are shown.