Figure 4

An over-expression of TRIP12 affects cell division by delaying mitotic entry. (A) The graph represents the percentage ± SEM of GFP-cells that overcome at least two cell divisions or die over a 48h-period. * and *** indicate a p value < 0.05 and < 0.001, respectively. (B) The graph represents the average duration ± SEM in interphase between two mitoses (white bars) and between mitosis and cell death (grey bars) in the different GFP positive cells. (C) Percentage of pHH3-Ser10 positive HelaS3 cells transfected with TRIP12(1-1992)-GFP or control CAT-GFP constructs and arrested by Ro-3306 treatment was assessed by flow cytometry after release in inhibitor free medium every 30 min for 2 h. The graph represents the mean ± SEM of three different experiments. * indicates a p value < 0.05. (D) Proportion of cells in G₂, prophase, early prometaphase, late prometaphase and metaphase in HelaS3 cells previously transfected with TRIP12(1-1992)-GFP or control CAT-GFP constructs was determined by immunofluorescence using anti-GFP (top panel) and anti-pHH3-Ser10 (middle panel) antibodies. Forty-eight hours after transfection, cells were arrested in G₂ phase by a Ro-3306 treatment and released in inhibitor free medium for 30 min. GFP-positive cells that were negative for pHH3-Ser10 staining were considered in G₂ phase. DNA was counterstained with DAPI. The graph represents the percentage of cells in the different phases expressed as mean ± SEM obtained from a minimum of 300 cells of three different experiments. ** indicates a p value < 0.01.