Figure 2.
Figure 2. Haspin inhibitors influence the maintenance of Aurora B activity toward centromeric targets. (A) Nocodazole-arrested U2OS cells were obtained as in Fig. 1 B, then kinase inhibitors and 20 µM MG132 were added for 1 h in the presence (MCAK and Aurora B) or absence (CENP-AS7ph and H3S10ph) of 0.33 µM nocodazole. Mitotic Aurora B localization, the centromeric staining intensity of MCAK and CENP-AS7ph, and H3S10ph intensity on chromosomes were classified for at least 100 cells in each condition in one experiment by immunofluorescence microscopy. Similar results were obtained in duplicate experiments. (B and C) Example images of cells treated as in A. Bars, 5 µm. (D) The intensities of CENP-AS7ph and H3S10ph on mitotic chromosomes in each condition were quantified (n = 6–14 cells). Results are expressed as a ratio to centromeric autoantigen staining intensity at the same centromeres. Means + SD are shown (error bars); ***, P < 0.001 vs. DMSO.

Haspin inhibitors influence the maintenance of Aurora B activity toward centromeric targets. (A) Nocodazole-arrested U2OS cells were obtained as in Fig. 1 B, then kinase inhibitors and 20 µM MG132 were added for 1 h in the presence (MCAK and Aurora B) or absence (CENP-AS7ph and H3S10ph) of 0.33 µM nocodazole. Mitotic Aurora B localization, the centromeric staining intensity of MCAK and CENP-AS7ph, and H3S10ph intensity on chromosomes were classified for at least 100 cells in each condition in one experiment by immunofluorescence microscopy. Similar results were obtained in duplicate experiments. (B and C) Example images of cells treated as in A. Bars, 5 µm. (D) The intensities of CENP-AS7ph and H3S10ph on mitotic chromosomes in each condition were quantified (n = 6–14 cells). Results are expressed as a ratio to centromeric autoantigen staining intensity at the same centromeres. Means + SD are shown (error bars); ***, P < 0.001 vs. DMSO.

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