Figure 5

A TRIP12 depletion accelerates mitotic entry. (A) Expression of Trip12 mRNA was determined by RT-qPCR in HelaS3 cells transduced with 3 shRNAs directed against Trip12 mRNA (ShTRIP12) or scramble control shRNA (ShScr). β-Actin, Gapdh and Cyclophilin A mRNA level was used for normalization. Results are the mean ± SEM obtained from three different experiments and expressed as percentage of expression compared to ShScr control cells. ** indicates a p value < 0.01. (B) Expression of TRIP12 protein in HelaS3 ShTRIP12 and ShScr cells was determined by Western blot analysis. GAPDH protein level was used as loading control. Images were obtained from the same experiment and representative of three different experiments. (C) Percentage of cells in metaphase in TRIP12-depleted (ShTRIP12#1, #2 and #3) and control (ShScr) HelaS3 cells arrested by a Ro-3306 treatment and released in the cell cycle by medium replacement (t = 0) was determined by cell live microscopy. The graph represents the percentage of cells in metaphase phenotype expressed as mean ± SEM relatively to total number of cells. A cell was considered in metaphase phenotype when the equatorial plate was microscopically visualized. A representative cell in metaphase phenotype is indicated by the white arrow. The quantification was performed from 9 different fields from three different experiments representing an average of 650 analyzed cells. * and ** indicates a p value < 0.05 and 0.01, respectively. (D) Proportion of cells in prophase, early prometaphase, late prometaphase and metaphase in TRIP12-depleted (ShTRIP12#1) and control (ShScr) HelaS3 cells arrested by a Ro-3306 treatment and released in the cell cycle by medium replacement was determined by immunofluorescence using anti-pHH3-Ser10 antibody after 30 min. DNA was counterstained with DAPI. The graph represents the percentage of cells in the different steps of early mitosis expressed as mean ± SEM obtained from a minimum of 300 cells of three different experiments. * and ** indicate a p value < 0.05 and 0.01, respectively. (E) Percentage of pHH3-Ser10 positive cells in TRIP12-depleted (ShTRIP12) and control (ShScr) HelaS3 cells arrested in G₂ phase by a Ro-3306 treatment and released in the cell cycle by medium replacement was determined by flow cytometry at the indicated time. Three different ShTRIP12 (#1, #2 and #3) were used for these experiments. The results represent the mean ± SEM obtained from four different experiments. *, ** and *** indicate a p value < 0.05, 0.01 and 0.001, respectively.