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. 2016 Feb 10;7(13):15703–15724. doi: 10.18632/oncotarget.7310

Figure 2. Adenovirus increases the mitotic index in gemcitabine-treated cells and enhances aberrant passage through mitosis.

Figure 2

a and b. Cell cycle and mitotic index analysis in synchronised PT45 cells, stained with propidium iodide (for DNA-content analysis), a phospho-histone H3 antibody (for mitotic index analysis) and an E1A antibody (for identification of infected cells) and analysed by flow-cytometry. Dead cells, as identified from their incorporation of FVD, and cells in subG1 phase were excluded from the analysis. a. Cell-cycle distribution of non-infected or infected cells, shown as % cells in G1-phase, S-phase, G2-phase and mitosis (M). Only S-phase statistical significance is shown. b. Mitotic Index (% cells in mitosis). Error bars represent S.E.M. of 4 independent experiments. c. Immunofluorescence microscopy analysis of Aurora-A (green) and α-Tubulin (red) with nuclear DAPI staining (DNA, blue) in PT45 cells fixed at 48hpi using methanol. Left panel: Example images of bipolar and multipolar spindles in cells treated with gemcitabine. Scale bar: 5μm. Right panel: Quantification of spindle multipolarity, expressed as frequency (%), ≥ 150 mitotic cells/condition were counted. Error bars represent S.E.M of two independent experiments. d-g. Time-lapse microscopy 24-96hpi in PT45 cells stably expressing histone H2B-mCherry. Error bars represent S.E.M of 3 independent experiments. Numbers on images indicate time (in minutes). Scale bar: 20μm. d. Scatter plot showing the duration of mitosis, defined from the time of nuclear envelope breakdown until the time of sister chromatid separation. At least 100 mitotic cells were analysed/condition/study in 3 independent studies. e. Frequency (%) (top panel) and example images (bottom panels) of bipolar and multipolar anaphases. At least 100 mitotic cells were analysed/condition/study in 3 independent studies. f. Frequency (%) (top panel) and example images (bottom panels) of cytokinesis failure. At least 100 mitotic cells were analysed/condition/study in 3 independent studies. g. Frequency (%) (top panel) and example images (bottom panels) of mononucleated, micronucleated and multinucleated daughter cells. At least 150 daughter cells were analysed/condition/study in 3 independent studies. a-g. *.p<0.05 **.p<0.01, ***.p<0.001 (one-way ANOVA with Bonferroni's multiple comparison test)