Figure 3

TOP2A decreases in PTEN deficient cells and ectopic expression of PTEN restores TOP2A levels and rescues decatenation deficiencies.

(a) Western blot analysis. Top2a, Pten and p53 levels in MEF Pten^+/+, MEF Pten^−/− , MEF p53^−/− or MEF Pten^−/−p53^−/− cells were analyzed with western blotting. (b) Topoisormerase II activity assays. Nuclear extracts of Pten^+/+, Pten^−/−, p53^−/− and Pten^−/− p53^−/− MEF cells were assayed for the ability to decatenate kinetoplast DNA (kDNA) with 2 ug of total protein added to each reaction system. (c) Flow cytometry analysis of mitotic cells. Dual color flow cytometry analysis was performed with phospho-Ser10 histone 3 and PI staining of MEF Pten^+/+ or Pten^−/− cells. MEF Pten^−/− cells with restored Pten or Top2a after UV or ICRF-193 treatment. Quantification data represent 3 independent experiments ± SEM, One-Way ANOVA, n.s. no significance, ***p < 0.001. (d) Statistical analysis of PICH-bridge positive cells. Anaphase HCT116 PTEN^+/+ or HCT116 PTEN^−/− cells were counted and grouped based on presence or absence of PICH bridges. Quantification data of percentage of cells with or without PICH bridges represent 3 independent experiments ± SEM. n = 40, One-Way ANOVA, **p < 0.01, ***p < 0.001. (e) Statistical analysis of PICH-bridge positive cells. Staining of PICH ultra-fine bridges in HCT116 PTEN^+/+ or PTEN^−/− cells and HCT116 PTEN^−/− cells expressing ectopic PTEN. Quantification data represent 3 independent experiments ± SEM. n = 40, One-Way ANOVA, ***p < 0.001. (f) Statistical analysis of PICH bridge positive cells. Column chart illustrating the proportion of cells with and without PICH bridges in HCT116 PTEN^+/+ or PTEN^−/− cells and in HCT116 PTEN^−/− cells expressing ectopic TOP2A. Quantification data represent 3 independent experiments ± SEM. n = 40, One-Way ANOVA, ***p < 0.001.