Hela cells grown on glass coverslips were treated by C5M (100 nM) or water (control, Co) in different trials and then, fixed by formaldehyde 4% for 10 minutes at 37°C. Immunofluorescences were performed as described previously [40 (link)]. Coverslips were incubated with the antibodies directed against the following antigens: phospho-histone H3-Serine10 (Upstate, 1:2000); phospho-histone H3-Serine 28 (Upstate, 1:2000); aurora kinase B (Epitomics, 1:2000); rabbit anti-phospho-AMPK (Thr172) (Cell Signaling, 1:100), rabbit anti-phospho-aurora A/B/C (Cell Signaling, 1:100). Actin was stained by phalloidin-rhodamin (1 μg/ml). DNA was visualized with Hoechst 33342 (Sigma, 0.5 μg/ml). Images were collected with a ZEISS 710 Laser Scanning Confocal microscope with a 63×-immersion oil objective. Slices of 0.5 μm are shown.
Metaphases spreading were realized as follows: HeLa cells were incubated ON with either C5M (200 nM) or taxol (33 nM) and were then recovered by flushing. An osmotic choc was realized (calf serum diluted in water 1:5) for 13 min, at 37°C, then the same volume of fixative (mixture of EtOH and acetic acid (3:1)) was added. After 20 min of incubation at room temperature, cells were washed by fixative and stored at 4°C. Finally cells were spread on cold coverglass and DNA was stained with Hoechst.
Metaphases spreading were realized as follows: HeLa cells were incubated ON with either C5M (200 nM) or taxol (33 nM) and were then recovered by flushing. An osmotic choc was realized (calf serum diluted in water 1:5) for 13 min, at 37°C, then the same volume of fixative (mixture of EtOH and acetic acid (3:1)) was added. After 20 min of incubation at room temperature, cells were washed by fixative and stored at 4°C. Finally cells were spread on cold coverglass and DNA was stained with Hoechst.
Free full text: Click here
