Figure 4

Up-regulation of Par14 in the S and G2/M phases. A) Percentage cell cycle distribution. Human foreskin fibroblasts (HFF) were examined by DNA staining with propidium iodide followed by FACS analysis. Numbers denote percentage of cells in G0, G1, S and G2/M phase. Percentage of cells at G0 was fixed to 100 after serum deprivation. The arithmetic mean (X-mean), is an indicator of the fluorescence intensity i.e. the number of propidium iodide molecules bound to DNA. A value above 200 is considered as appropriate. The results show the average of five independent cell cycle synchronisation experiments. For each sample, 1 × 10⁴ cells were analysed B) qRT-PCR analysis of Par14 across the cell cycle with synchronised HFF. White columns represent the percentage mRNA expression, dark blue columns the percentage mRNA expression of cyclin B2. After 30–36 hrs of serum deprivation, cells in G0 were arrested at t = 0 h. HFF cells were re-stimulated with 20% FCS. G1 cells were harvested after t = 14 h, S phase cells after t = 20 h and G2/M cells after t = 24 hrs. mRNA levels of Par14 were normalised using snRNA U6. One-way ANOVA was performed using R. The bars represent the standard deviation from five independent cell cycle synchronisation and qRT-PCR experiments conducted. The asterisks on the bars indicate the statistical significance expressed in p-values (** = p ≤ 0.01; * = p ≤ 0.05). C) Translational up-regulation of protein across the cell cycle. Western blot analysis was done using αPPIase (1:1000) and β- actin (1:5000) antisera. 30 μg of protein for each cell lysate from the respective phase of the cell cycle was loaded. D) Densitometric analysis from three independent cell cycle synchronisation and western blot experiments of Par14 protein. Band signals were normalised with β- actin.