Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Phosphorylation of serine-10 of histone H3 shields modified lysine-9 selectively during mitosis.

doi: 10.1111/j.1365-2443.2009.01375.x

Figure Lengend Snippet: Figure 1 Fluctuation in the level of dimethylated H3K9 detected during mitosis. (A) Immunostaining of cultured cells for H3K9 methylation. (B) Immunostained cells arranged according to cell cycle progression. To improve antibody access, fixed cell samples were denatured using an acid solution before antibody incubation. See Fig. S1 in Supporting Information for pictures obtained when using a conventional immunostaining protocol. Although trimethylated H3K9 (H3K9me3) signals appear steady, dimethyl- ated H3K9 (H3K9me2) signals oscillate during the cell cycle in NIH3T3 cells. (C) Eight-cell stage mouse embryos. Arrows in A and C indicate bundles of mitotic chromosomes possessing H3K9me3 signals but not H3K9me2 signals. Nuclei were counter- stained with 4¢,6-diamidino-2-phenylindole (DAPI; blue). (D-E) Western blot analysis for estimating the level of H3K9me2 in cells arrested in (pro)metaphase using colcemid (D) or nocodazole (E). In (E) the Aur-B inhibitor ZM447439 was used in conjunction with nocodazole (see Fig. 3). H3S10ph, phosphorylated histone H3 serine 10; H3, histone H3; Ana-telophase, anaphase and telophase.

Article Snippet: Cell cycle arrest and chemical treatments For ZM447439 (Tocris Bioscience, Ellisville, MO, USA) treatment (Ditchfield et al. 2003), cells were grown in culture media containing 2 lM ZM447439 for 1 h at 37 C, washed with phosphate-buffered saline (PBS), andfixed with4% formaldehyde solution (Sigma Aldrich, St Louis, MO, USA).

Techniques: Immunostaining, Cell Culture, Methylation, Incubation, Staining, Western Blot

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Phosphorylation of serine-10 of histone H3 shields modified lysine-9 selectively during mitosis.

doi: 10.1111/j.1365-2443.2009.01375.x

Figure Lengend Snippet: Figure 3 Depletion of H3S10 phosphorylation is associated with the emergence of H3K9me2 signals on mitotic chromosomes. (A) Phosphorylated H3S10 (H3S10ph) as a mitotic index. Cells were doubly stained for H3S10ph (red) and H3K9me2 (green). H3S10ph signals emerge during G2 phase in contrast to the signals for H3K9me2. (B) Depletion of H3S10ph by treatment with the Aurora B Kinase (Aur-B) inhibitor ZM447439. ZM447439 treatment causes NIH3T3 cells to arrest at prometaphase; these cells lose H3S10ph signals and gain H3K9me2 signals (arrows in b). (C) Diffuse H3K9me2 signals in ZM447439-treated chromo- somes. Diffuse H3K9me2 signals are seen over the entire mitotic chromosomes that lack H3S10ph signals. ZM47439-treated cells were doubly stained for H3K9me1 (red) and H3K9me2 (green) in c and f. (D) Transient expression of the GFP-AurK106R domi- nant negative mutant protein. In GFP-Aur-BK106R-expressing NIH3T3 cells, H3S10ph signals are greatly reduced (b) and solid H3K9me2 signals reappear on mitotic chromosomes (d). GFP-Aur-B protein was also expressed as a control (a and c). Nuclei were counterstained with DAPI (blue).

Article Snippet: Cell cycle arrest and chemical treatments For ZM447439 (Tocris Bioscience, Ellisville, MO, USA) treatment (Ditchfield et al. 2003), cells were grown in culture media containing 2 lM ZM447439 for 1 h at 37 C, washed with phosphate-buffered saline (PBS), andfixed with4% formaldehyde solution (Sigma Aldrich, St Louis, MO, USA).

Techniques: Phospho-proteomics, Staining, Expressing, Mutagenesis, Control

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: Phosphorylation of serine-10 of histone H3 shields modified lysine-9 selectively during mitosis.

doi: 10.1111/j.1365-2443.2009.01375.x

Figure Lengend Snippet: Figure 5 HP1 proteins localize to pericentric regions of H3S10-dephosphorylated mitotic chromosomes. (A) Localization of DsRed2-HP1 isoforms during mitosis. Cells stably expressing DsRed2-tagged HP1a, -HP1b and -HP1c proteins are lined up according to mitotic progression. HP1b is associated with chromatin during late mitosis (d–g), whereas HP1a and HP1c associate with other mitotic structures, such as microtubules of the central spindles (k, arrowhead) and the midbody (arrows in l and r). (B) DsRed2-HP1 proteins in ZM447439-treated mitotic chromosomes. DsRed2-HP1 produces punctuate signals at pericentric regions of prometaphase chromosomes in ZM447439-treated cells but not in DMSO-treated control cells (DMSO). (C) Distribution of endogenous HP1b proteins (green) and H3K9me2 (a) and H3K9me3 signals (e) in ZM447439-treated mitotic chromosomes. Nuclei were counterstained with DAPI (blue).

Article Snippet: Cell cycle arrest and chemical treatments For ZM447439 (Tocris Bioscience, Ellisville, MO, USA) treatment (Ditchfield et al. 2003), cells were grown in culture media containing 2 lM ZM447439 for 1 h at 37 C, washed with phosphate-buffered saline (PBS), andfixed with4% formaldehyde solution (Sigma Aldrich, St Louis, MO, USA).

Techniques: Stable Transfection, Expressing, Control

Journal: BMC biology

Article Title: Evolutionary conserved relocation of chromatin remodeling complexes to the mitotic apparatus.

doi: 10.1186/s12915-022-01365-5

Figure Lengend Snippet: Fig. 7 Inhibition of Aurora B kinase activity with ZM447439 and Barasertib affected the localization of BAF53a and Tip60 to the midbody. A Treatment with ZM447439: from left to right: DAPI (blue), anti- α-tubulin (green), tested protein (red), and merge. Anti-MKLP1 and anti-Aurora B immunostaining were used as positive and negative controls, respectively. Scale bar = 10 μm. The localization pattern of BAF53a and Tip60, as well as that of MKLP1 were affected, while no effect was seen for SRCAP and Aurora B. B The effects found with ZM447439 treatment are summarized in the graph. At least 300 telophases were scored in three independent experiments for both treated cells and control HeLa cells. C To test the effectiveness of ZM447439 inhibition, phosphorylation of the histone H3 (target of Aurora B kinase) during mitosis was evaluated; α-tubulin was used as loading control. D The effects found with Barasertib treatment are summarized in the graph. At least 200 telophases were scored in two independent experiments for both treated cells and control HeLa cells. A slight effect on Aurora localization was seen at the limit of significance values. E To test the effectiveness of Barasertib inhibition, phosphorylation of the histone H3 (target of Aurora B kinase) during mitosis was evaluated; α-tubulin was used as loading control. *=P<0.05, **<=P<0.005, ***=P<0.0005

Article Snippet: Asynchronous HeLa cells were treated for 35 min with the Aurora B inhibitor ZM447439 (SignalChem, A31901-01) and Barasertib (AZD1152-HQPA, Selleckchem, S1147) at a final concentration of 5 μM and 200 nM, respectively, and then fixed and stained.

Techniques: Inhibition, Activity Assay, Immunostaining, Control, Phospho-proteomics