Fig. 2.
p110 CUX1 expression enables bipolar mitoses in newly formed tetraploid cells. (A) Proliferation curves of NMuMG/vector (bold) and NMuMG/CUX1 (dashed) cells, with (gray) or without (black) a 12-h blebbistatin pulse treatment. Cells were counted until they ceased proliferation because of contact inhibition. (B) Percentage of apoptotic cells was measured 48 h after blebbistatin wash-off, according to Annexin V staining and FACS analysis. (C and D) Outcome of cell divisions after a blebbistatin wash-off was determined by time-lapse microscopy using frames taken every 5 min. Multipolar divisions displayed a multipolar anaphase giving rise to three or four daughter cells (Movie S1) or a tripolar anaphase followed by cytokinesis failure resulting in two cells, one of which with two nuclei (Movie S2). Neighboring mononucleated cells were used as controls. (Scale bars, 25 μm.) (E) U2OS and MCF10A cells expressing p110 CUX1 were treated with blebbistatin, and the fate of binucleated cells was determined by time-lapse microscopy. (F) NMuMG/CUX1 were pulse-treated with blebbistatin and imaged by time-lapse microscopy with increasing concentrations of MPS1-IN-1. The outcome of cell division and the duration of mitosis were scored for mono- and binucleated cells. (G) U2OS/vector cells were pulse-treated 8 h with blebbistatin. Sixteen hours later, cells were imaged by time-lapse microscopy, during which 10 μM MG132 was added for 90 min and washed away. The fate of binucleated cells entering mitosis during or just before MG132 treatment (Movie S5) was examined. Neighboring binucleated cells dividing without being exposed to MG132 were used as control (Movie S4). Alternatively, cells were fixed 1 h after MG132 treatment, and the metaphase spindle configuration was determined using centrin 3 and α-tubulin staining (Fig. S1 I–K). *P < 0.005; **P < 0.0002; ***P ≤ 1.2 × 10−5. Black bars, % bipolar division. Gray bars, % multipolar division.