Figure 1
From: PTEN stabilizes TOP2A and regulates the DNA decatenation
Failure of decatenation in Pten deficient cells results in the formation of ultra-fine bridges in anaphase cells.
(a) Immunofluorescence of PICH-coated ultra-fine bridges. Pten+/+ or Pten−/− MEFs were stained with the α-PICH antibody (green). Representative anaphase cells are shown with and without PICH-coated bridges. (b) Statistical analysis of a. Anaphase Pten+/+ or Pten−/− MEFs were counted and grouped based on presence or absence of PICH bridges. Quantification data to determine the percentage of cells with or without PICH bridges are representative of 3 independent experiments ± SEM. n = 40, One-Way ANOVA, **p < 0.01. (c) Immunofluorescence of PICH-coated ultra-fine bridges and centromeres. HCT116 PTEN+/+ and PTEN−/− cells were stained for PICH (green), CREST (red) and DNA (DAPI). Cells with no bridges (left); bridges with CREST foci (middle); and bridges without CREST foci (right); (d) Statistical analysis of c. Stacked column chart illustrating the proportion of cells with PICH bridges positive or negative for CREST staining. Quantification data represent 3 independent experiments ± SEM. n = 40, One-Way ANOVA, **p < 0.01. (e) Immunofluorescence of PICH-coated ultra-fine bridges. HCT116 PTEN+/+ or PTEN−/− cells were treated with ICRF-193 prior to PICH (red) immunofluorescent staining. Cells which are illustrated are anaphase cells with no, one or multiple PICH-coated bridges. (f) Statistical analysis of e. Column chart illustrating the proportion of ICRF-193 treated and untreated HCT116 cells with and without PICH bridges. Quantification data represent 3 independent experiments ± SEM. n = 40, One-Way ANOVA, **p < 0.01, ***p < 0.001. (g) Flow cytometry analysis of mitotic cells. MEF cells were treated with UV or ICRF-193 prior to dual-color flow cytometry analysis with phospho-Ser10 histone 3 and PI (propidium iodide) staining. Quantification data represent 3 independent experiments ± SEM. One-Way ANOVA, *p < 0.05, **p < 0.01. (h) Metaphase spread followed by centromeric FISH. Shown are representative images of MEF metaphase spreads after 1 μM ICRF-193 treatment for 2 h with normally condensed chromosomes, undercondensed chromosomes and entangled chromosomes. (i) Statistical analysis of (h). Column chart showing the proportion of ICRF-193 cells with normal, undercondensed or entangled chromosomes. Quantification data represent 3 independent experiments ± SEM. n = 50, One-Way ANOVA; n.s. no significance, ***p < 0.001.
