Figure 6

A shortened duration of DNA replication explains the accelerated mitotic entry of TRIP12-depleted cells. (A) TRIP12, WEE1, CDC25C and CDK1 protein and P-Try15-CDK1 phosphorylation levels in nuclear and cytosolic fractions of TRIP12-depleted and control HelaS3 cells after a Ro-3306 treatment were determined by Western blot analysis. 4EBP1 and panH3 protein levels were used as loading and purity controls. Images were obtained from the same experiment and representative of three different experiments. The asterisk and the arrow indicate the position of phosphorylated and non-phosphorylated forms of CDK1, respectively. (B) CYCLIN A level in nuclear and cytosolic fractions of TRIP12-depleted and control HelaS3 cells after Ro-3306 treatment was determined by Western blot analysis. 4EBP1 and panH3 protein levels were used as loading and purity controls. Images were obtained from the same experiment and representative of three different experiments. (C) CYCLIN A nuclear presence in TRIP12-depleted and control cells after RO-3306 treatment was determined by immunofluorescence. The image illustrates representative cells with nuclear CYCLIN A (white arrows) or not. The graph represents the percentage of cells expressed as mean ± SEM obtained from three different experiments. Percentage of cells in G₂ phase (white bars), late S (light grey bars) and early S (dark grey bars) was determined by immunofluorescence using criteria defined in Fig. 2C. The bars represent the mean obtained from three different experiments. * and ** indicate a p value < 0.05 and 0.01, respectively. (D) Percentage of EDU positive cells in TRIP12-depleted (ShTRIP12) and control (ShScr) HelaS3 cells arrested in early S phase by a double thymidine block treatment and released in the cell cycle was determined by EDU incorporation at the indicated time. The graph represents the mean ± SEM obtained from a minimum of 2500 cells for each indicated time of three different experiments. *, ** and *** indicate a p value < 0.05, 0.01 and 0.001, respectively. (E) Percentage of early S and late S cells in EDU-positive TRIP12-depleted (ShTRIP12) and control (ShScr) HelaS3 cells was determined by immunofluorescence using criteria defined in Fig. 2C. The graph represents the mean ± SEM obtained from a minimum of 500 EDU-positive cells of three different experiments. * and ** indicate a p value < 0.05 and 0.01, respectively.