γH2AX foci induced by ionizing radiation are absent from HP1α-staining nuclear domains.

<p>MCF7 cells were fixed 30 minutes after X-irradiation (2Gy, panels a–l & p–r) and were processed for immunofluorescence for γH2AX (green) and either HP1α or H3K9Me3 (red). Panels m–o, non-irradiated cells. DNA was stained with DAPI. Panels a, d, g, j, m & p, γH2AX; panels b, e, h, k & n, HP1α; Panel q, H3K9Me3; panels c, f, I, l, o & r merged images. Overlapping red and green signals appear yellow. The top row shows a group of cells with typical appearance. Individual nuclei are shown magnified in rows 2–6. For the nuclei shown in rows 2–4, line traces were generated (shown on the right) with the line drawn through the brightest HP1α regions. For γH2AX, HP1α and H3K9Me3, images were first adjusted using levels such that fainter interfocal nuclear fluorescence was not included.</p

G₁ and S-phase H2AX phosphorylation.

<p>MCF7 cells were serum starved (0.05% FCS) for 24 hours before release into medium containing 20% FCS. Cells were fixed at different times such that fixed cell populations were predominantly in G₁, early S (S-E) or late S-phase (S-L). (6.5 hr, 24 hr and 38 hr respectively). HU or cisplatin were added to the cells, to 2 mM or 50 µM respectively, at the indicated times prior to fixation. Irradiated cells were fixed 30 minutes after irradiation.</p

Distribution of X-ray induced γH2AX foci in relation to HP1α staining during G₁ versus late S-phase.

<p>MCF7 cells were serum starved (0.05% FCS) for 24 hours before release into medium containing 20% FCS. Cells were X-irradiated (2Gy) at different times and fixed 15 minutes later, such that fixed cell populations were predominantly in G₁ or late S-phase. (6.5 hr, 38 hr respectively). Correlation between HP1α and H2AXγ signals was determined by eye from overlaid immunofluorescence images. Mean numbers of nuclei exhibiting the described coincidence of HP1α and H2AXγ signals were derived from at least two fields containing in excess of 100 nuclei.</p>*<p>p<0.001 (t-test).</p

Replication stress can induce phosphorylation of histone H2AX in heterochromatin.

<p>Panels a–l, subconfluent asynchronous MCF7 cells were exposed to hydroxyurea (2 mM) for one hour immediately prior to fixation and processing for γH2AX (green) and HP1α (red) immunofluorescence. Panels a–c, representative nuclei displaying non-S phase, early to mid S-phase (S-E) and late S-phase (S-L) γH2AX staining respectively. Panels d–f & g–I, single S-L nuclei; j–l, single S-E nucleus. Panels d, g, j, γH2AX; e, h, k, HP1α; f, i, l, merged γH2AX/HP1α images. Line traces are presented on the right. Lines were drawn across the nucleus through heterochromatic (HP1α staining) regions in each case, including the DAPI channel. Panels m–r, subconfluent MCF7 cells were exposed to cisplatin (50 µM) for one hour, 38 hours after release from serum starvation. Cells were fixed immediately after cisplatin treatment and processed for γH2AX immunofluorescence. Panels m & p, γH2AX; n & q, DAPI; o & r merged γH2AX (green)/DAPI (red) images.</p

γH2AX foci generated by etoposide treatment do not appear in HP1α-staining regions in MCF7 cells.

<p>MCF7 cells were incubated in medium containing etoposide and processed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001057#pone-0001057-g001" target="_blank">Figure 1</a>. Panels a–c, two hour exposure to 5 µM etoposide. Panels d–i, 24 hour exposure to 1 µM etoposide. Panels j–l, untreated cells. Cells were fixed immediately after etoposide treatment.</p

Spatial relationship between γH2AX foci, HP1α-staining heterochromatic regions and nucleoli.

<p>Panel b–d and f–h were obtained from a single nucleus fixed 30 minutes after X-irradiation (2Gy) and processed for γH2AX and HP1α immunofluorescence. Panels, a & e were obtained from a separate irradiated nucleus processed for nucleolin and γH2AX immunofluorescence. Panel a, DAPI; b, γH2AX; c, HP1α; d, DAPI; e, merged DAPI (blue) and nucleolin (red), γH2AX (green) images; f, γH2AX (green)/DAPI (blue); g, HP1α (red)/DAPI (blue); h, γH2AX/HP1α/DAPI. In panels e–h, the red and green chanels were reduced to binary images, retaining as much detail as possible, before overlaying on the DAPI image. DAPI staining was carried out under optimum conditions to reveal nuclear structure.</p

Distribution of γH2AX foci in relation to HP1α staining in MCF7 cells treated with DNA damaging agents.

<p>Correlation between HP1α and H2AXγ signals was determined by eye from overlaid immunofluorescence images. In each case more than 50 nuclei were scored from 2 separate images.</p