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. 2017 Jan 10;3:16049. doi: 10.1038/celldisc.2016.49

Figure 4.

Figure 4

Aurora-A modulates H3T3-ph in a chromosomal passenger complex (CPC)-independent manner. (a) Diagram showing the immunoblotting assay performed to examine H3T3-ph levels. Cells were synchronized by releasing from double thymidine block, M phase cells were collected by shake-off 9 h after release and the attached cells after shaking-off were regarded to be cells in G2 phase. (b) H3T3-ph and H3S10-ph level were monitored using immunoblotting in synchronized Hela cells treated with Aurora-A (50 nM), Aurora-B (100 nM) and Plk1 (100 nM) inhibitors. (c, d) Quantification of the level of H3T3-ph (c) and H3S10ph (d) in b. Three independent experiments were performed. (e) Tethering CPC at centromeres failed to rescue H3T3-ph upon Aurora-A inhibition. Hela cells transfected with CB-INCENP were treated with dimethyl sulfoxide (DMSO) or MLN8237 for 2 h. H3T3-ph was monitored with immunostaining. (f) H3T3-ph in e is quantified. n=60, three independent experiments were performed, Values represent as mean±s.e.m. ***P<0.001 (Student’s t-test), DAPI, 4,6-diamidino-2-phenylindol; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NS, not significant. Scale bar: 10 μm.