Increased ploidy and centrosome amplification in Bcl-3-depleted cells. A) Histogram showing HeLa cells stably expressing control (black bars) or Bcl-3 (white bars) shRNAs were treated with colcemide and subjected to a karyotype analysis. Careful examination of fifty karyograms was perfomed and chromosome counts obtained. B) Table showing percentage and standard deviation of cell populations in G1, S and G2/M examined by flow cytometry analysis. C) HeLa cells stably expressing control or Bcl-3 shRNAs stained with an anti- gamma tubulin antibody and visualized using fluorescence microscopy. The graph shows percentage of control (black bars) or Bcl-3-depleted (white bars) cells with 2, 3 or more than three centrosomes at mitosis. Two hundred and fifty fields were scored in three independent experiments. * p = 0.02; **p = 0.04 by Student's T test. Inset: representative image. Arrows show centrosomes. D) Western blot analyses of cell with downregulated Bcl-3 levels. E) Effect of the expression of a Bcl3 shRNA in the number of HeLa cells at the indicated cell cycle phase (number of cells/4000 cells). The cells were transfected with a plasmid that direct the expression of a Bcl3 or a control shRNA and, simultaneously, an Enhanced Green Fluorescent Protein as a marker. After 48 hours, the cells were stained with an antibody directed toward γ-tubulin, a Cy5-marked secondary antibody and DAPI. Phases were scored only from green cells and results were pooled from three separate experiments. P: Prometaphase, M: Metaphase, A: Anaphase, T: Telophase, C: Cytokinesis.