Supplmentary Information 2005-04-04743

Supplmentary Information 2005-04-04743

Corresponding Author: Jan-Michael Peters

Title: Histone H3 serine-10 phosphorylation by Aurora-B causes HP1 dissociation from heterochromatin.

Supplementary Figure 1

Characterization of MCA2 and MCA6.

a, MCA2 and MCA6 react specifically with a mitosis specific modification on histone H3. Total cell extracts from cells arrested in S phase by hydroxyurea treatment (I) or in mitosis by nocodaozole treatment (M) were resolved by 14% SDS- PAGE, transferred onto a PVDF membrane, stained with Ponceau S (left panel), and analyzed in immunoblots with MCA2 or MCA6 sera (right panel). Bands corresponding to core histones (H2A, H2B, H3 and H4) are indicated.

b, IFM analysis of MCA2 and MCA6. Logarithmically proliferating HeLa cells were fixed and stained with the indicated sera (green), and DNA was counterstained with DAPI (blue). Representative cells in interphase, prophase, prometaphase, metaphase and anaphase are shown. Scale bar: 10 mm.

c, Peptide competition of MCA1 staining. The indicated peptides were preincubated with antibodies at a final concentration of 30 mM for 60 min before being used in IFM experiments.

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Supplementary Figure 2

The MCA1 epitope is conserved among species.

Rat kangaroo PtK2 cells and Drosophila S2 cells were treated with a hypotonic buffer, cytospun onto glass slides, and processed for IFM analysis. Note that in both cell lines, interphase cells did not react with MCA1 serum, but in mitotic cells pericentric loci were specifically stained, as in HeLa cells or in MEFs.

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Supplementary Figure 3

H3 phosphopeptides do not compete with MCA1 reactivity. 30 mM of the indicated H3 peptides were preincubated with antibodies for 60 min and then used in IFM experiments. Under these conditions, H3S10ph and H3S28ph peptides could specifically abolish staining of chromosomes with antibodies to these two sites.

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Supplementary Figure 4

Localization of HP1a in interphase and at different stages of mitosis. Logarithmically proliferating HeLa cells were incubated with 100 nM Hesperadin or with the solvent 0.1% DMSO for 30 min and stained with HP1a antibodies (a) and MCA1 serum (red in b). DNA was counterstained by DAPI (blue in b). Scale bar: 10 mm.

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Supplementary Figure 5

Inhibition of Aurora-B also affects association of HP1b and HP1g with mitotic chromosomes. HeLa cells grown on coverslips were incubated with 100 nM Hesperadin or with the solvent, 0.1% DMSO, for 30 min and processed for IFM analysis using antibodies to HP1a, HP1b or HP1g. Representative cells in prometaphase are shown (upper panels). The fluorescence intensities of HP1a (n=19), HP1b (n=18) or HP1g (n=20) on chromosomes were quantified, and the resulting values (average ± stdv.) of cells that had been treated with Hesperadin are shown relative to those of DMSO treated cells.

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Supplementary Figure 6

Depletion of Aurora-A and -B. 48 hr after the transfection of indicated siRNAs, cell extracts were analyzed by immunoblotting for Aurora-A and Aurora-B.

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Supplementary Table

Characterization of all six MCA sera.

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