Supplemental Material

This article contains the following supporting material:

• Supplementary Movie 1 - Unirradiated Interphase Indian Muntjac Fibroblast Immunofluorescently Labeled with anti-γ-H2AX.

  High-resolution (100x) 360° rotation of an unirradiated IM fibroblast nucleus counterstained with DAPI (top left panel) and immunofluorescently labeled with anti-γ-H2AX (top right panel). A merged image is included (bottom left), where the DNA is pseudo-colored red and γ-H2AX green. The acquired z-series was subjected to deconvolution as described in Materials and Methods and a 3D rotation was generated in Metamorph. This image was acquired to best depict the γ-H2AX foci. Note that the predominant proportion of the γ-H2AX foci are small with only a few larger amorphous foci are present.

• Supplementary Movie 2 - Unirradiated Mitotic Indian Muntjac Fibroblast Immunofluorescently Labeled with anti-γ-H2AX.

  High-resolution (100x) 360° rotation of an unirradiated early prometaphase IM fibroblast nucleus counterstained with DAPI (top left panel) and immunofluorescently labeled with anti-γ-H2AX (top right panel). A merged image is included (bottom left), where the DNA is pseudo-colored red and γ-H2AX green. The acquired z-series was subjected to deconvolution as described in Materials and Methods and a 3D rotation was generated in Metamorph. This image was acquired to best depict the γ-H2AX foci. Note that the predominant proportion of the γ-H2AX foci are small with only a few larger amorphous foci are present.

• Supplementary Movie 3 - Irradiated Interphase Indian Muntjac Fibroblast Immunofluorescently Labeled with anti-γ-H2AX.

  High-resolution (100x) 360° rotation of an irradiated IM fibroblast nucleus counterstained with DAPI (top left panel) and immunofluorescently labeled with anti-γ-H2AX (top right panel). Cells were irradiated with 2Gy and permitted to recover for 30min. A merged image is included (bottom left), where the DNA is pseudo-colored red and γ-H2AX green. The acquired z-series was subjected to deconvolution as described in Materials and Methods and a 3D rotation was generated in Metamorph. This image was acquired to best depict the γ-H2AX foci. Note the dramatic increase in the number of large γ-H2AX, while small γ-H2AX foci are still present.

• Supplementary Movie 4 - Irradiated Mitotic Indian Muntjac Fibroblast Immunofluorescently Labeled with anti-γ-H2AX.

  High-resolution (100x) 360° rotation of an irradiated prophase IM fibroblast nucleus counterstained with DAPI (top left panel) and immunofluorescently labeled with anti-γ-H2AX (top right panel). Cells were irradiated with 2Gy and permitted to recover for 30min. A merged image is included (bottom left), where the DNA is pseudo-colored red and γ-H2AX green. The acquired z-series was subjected to deconvolution as described in Materials and Methods and a 3D rotation was generated in Metamorph. This image was acquired to best depict the γ-H2AX foci. Note the dramatic increase in the number of large γ-H2AX, while small γ-H2AX foci are still present.

• Supplementary Movie 5 - The Spatial Relationship Between γ-H2AX Foci and 53BP1 in Unirradiated Human Cells.

  High-resolution (100x) 360° rotation of an unirradiated human SK-N-SH nucleus counterstained with DAPI and immunofluorescently labeled with anti-γ-H2AX (red) and anti-53BP1 (green). The acquired z-series was subjected to deconvolution as described in Materials and Methods and a 3D rotation was generated in Metamorph. Only the first frame contains the DAPI channel (white) and is for reference purposes. A cell containing as few large γ-H2AX repair foci was purposely selected to maximize channel detail and permit spatial comparisons between the small foci.

• Supplementary Movie 6 - The Spatial Relationship Between γ-H2AX Foci and BRCA1 in Unirradiated Human Cells.

  High-resolution (100x) 360° rotation of an unirradiated human SK-N-SH nucleus counterstained with DAPI and immunofluorescently labeled with anti-γ-H2AX (red) and anti-BRCA1 (green). The acquired z-series was subjected to deconvolution as described in Materials and Methods and a 3D rotation was generated in Metamorph. Only the first frame contains the DAPI channel (white) and is for reference purposes. A cell containing as few large γ-H2AX repair foci was purposely selected to maximize channel detail and permit spatial comparisons between the small foci.

• Supplementary Movie 7 - The Spatial Relationship Between γ-H2AX Foci and Mre11 in Unirradiated Human Cells.

  High-resolution (100x) 360° rotation of an unirradiated human SK-N-SH nucleus counterstained with DAPI and immunofluorescently labeled with anti-γ-H2AX (red) and anti-Mre11 (green). The acquired z-series was subjected to deconvolution as described in Materials and Methods and a 3D rotation was generated in Metamorph. Only the first frame contains the DAPI channel (white) and is for reference purposes. A cell containing as few large γ-H2AX repair foci was purposely selected to maximize channel detail and permit spatial comparisons between the small foci.

• Supplementary Movie 8 - The Spatial Relationship Between γ-H2AX Foci and Rad51 in Unirradiated Human Cells.

  High-resolution (100x) 360° rotation of an unirradiated human SK-N-SH nucleus counterstained with DAPI and immunofluorescently labeled with anti-γ-H2AX (red) and anti-Rad51 (green). The acquired z-series was subjected to deconvolution as described in Materials and Methods and a 3D rotation was generated in Metamorph. Only the first frame contains the DAPI channel (white) and is for reference purposes. A cell containing as few large γ-H2AX repair foci was purposely selected to maximize channel detail and permit spatial comparisons between the small foci.

• Supplementary Movie 9 - The Spatial Relationship Between γ-H2AX Foci and NBS1 in Unirradiated Human Cells.

  High-resolution (100x) 360° rotation of an unirradiated human SK-N-SH nucleus counterstained with DAPI and immunofluorescently labeled with anti-γ-H2AX (red) and anti-NBS1 (green). The acquired z-series was subjected to deconvolution as described in Materials and Methods and a 3D rotation was generated in Metamorph. Only the first frame contains the DAPI channel (white) and is for reference purposes. A cell containing as few large γ-H2AX repair foci was purposely selected to maximize channel detail and permit spatial comparisons between the small foci.

• Supplementary Movie 10 - The Spatial Relationship Between γ-H2AX Foci and ATM in Unirradiated Human Cells.

  High-resolution (100x) 360° rotation of an unirradiated human SK-N-SH nucleus counterstained with DAPI and immunofluorescently labeled with anti-γ-H2AX (red) and anti-ATM (green). The acquired z-series was subjected to deconvolution as described in Materials and Methods and a 3D rotation was generated in Metamorph. Only the first frame contains the DAPI channel (white) and is for reference purposes. A cell containing as few large γ-H2AX repair foci was purposely selected to maximize channel detail and permit spatial comparisons between the small foci.

• Supplemental Figure 1 - Localization of Three Different Anti-γ-H2AX Antibodies

  Asynchronous HeLa cells were paraformaldehyde-fixed, permeabilized, immunofluorescently labeled with each of the three anti-γ-H2AX (indicated at top - green) and counterstained with DAPI (red). Representative high-resolution (100x) DIM images are shown for the DAPI and γ-H2AX channels (single plane), a merge (DAPI and γ-H2AX channels) and a 3D projection of each nucleus (Proj.). Note that only JBW301 and ab2893 identify both large and small γ-H2AX foci and in approximately similar numbers, while ab11174 does not appear to recognize γ-H2AX in situ. Scale bar represents 2μm.

• Supplemental Figure 2 - The Specificities of Anti-&gamma:-H2AX Antibodies

  Depicted here are representative DIM images (100x) of 10T1/2 cells treated with Saponin with (CIP) or without (control) calf alkaline intestinal phosphatase (CIP) and immunofluorescently labeled with both anti-PhosS10 and anti-γ-H2AX (JBW301). The prophase 10T1/2 fibroblast CIP-treated cell was specifically selected, as it should normally exhibit a dramatic increase in PhosS10 immuno-staining over interphase cells, particularly within pericentromeric heterochromatin. Identical exposure times were used in the top two rows for all three channels with a 5-fold increase in exposure time (long exposure) for the anti-PhosS10 and anti-γ-H2AX channels shown in the third row. Scale bar represents 2μm.

• Supplemental Figure 3 - In Situ Indirect Immunofluorescent Peptide Competition Assay

  Asynchronous HeLa cells were immunofluorescently labeled with anti-γ-H2AX, JBW301 (A) and ab2893 (B), that were pre-incubated with either a competing H2AX peptide phosphorylated at the corresponding serine residue (H2AXp) or a similar non-phosphorylated H2AX peptide (H2AXnp). Depicted here are representative high-resolution (100x) DIM images. Increased (5-fold) exposure times were employed to image the γ-H2AX channel of the H2AXp experiments for both antibody treatments (i.e. JBW301 or ab2983). Note the absence of total γ-H2AX nuclear signal in the JBW301 H2AXp column (A) and the weak background staining pattern in the ab2893 H2AXp column (B) that is present in approximately equal amounts within both the cytoplasm and the nucleoplasm. For reference purposes a 3D projection (Proj) has been included as presents channel information of each nucleus. Scale bars represent 3μm

• Supplemental Figure 4 - Spatial Localization of Phospho-ATM in IM Fibroblasts

  Asynchronous IM Fibroblasts were paraformaldehyde-fixed, permeabilized, immunofluorescently labeled with anti-phospho-ATM (serine 1981 [p-ATM]) and counterstained with DAPI. Depicted here are high-resolution (100x) representative images at interphase, prophase, metaphase and anaphase that were subjected to image deconvolution. Single planes are presented for the DAPI (red), p-ATM (green) and Merge images, while the signals through each nucleus is presented as a 3D projection (Proj). Scale bars represent 3μm.

• Supplemental Figure 5 - The sensitivity of H2A.X phosphorylation in GM38 cells to inhibition by caffeine.

  Examples of fields of interphase GM38 cells stained for the presence of γ-H2AX following a four hour treatment with increasing concentrations of caffeine. The label indicates the concentration of caffeine that the cells were treated with. The inset in the bottom left of each panel is the corresponding DAPI image, which serves as an indicator for the location of individual nuclei, including those containing low levels of γ-H2AX .

• Supplemental Figure 6 - Qualitative Increases in the Abundance of γ-H2AX in Mitotically-enriched Cells

  Acid-solubilized proteins were isolated from nuclear preparations of asynchronous (A) or mitotically-enriched (M) populations generated by nocodazole treatment. Nine independent immunoblot assays were performed in triplicate and a single representative blot is shown for each cell line (indicated at top) — qualitative comparisons cannot be made between cell lines as each blot was individually optimized for each cell line. The CPTS-stained PVDF membrane has been included to confirm similar loading between A and M populations for each cell line. Note that for the first eight cell lines (HeLa through to GM38) a marked difference in the abundance of γ-H2AX occurs, with the mitotically-enriched population exhibiting the greatest signal intensity. For AT2BE (ATM-defective), nearly identical signal intensities of γ-H2AX occurs in the asynchronous and mitotically-enriched population, despite a 3.69-fold increase in the mitotic population (see Supplementary Table 1).

• Supplemental Figure 7 - Normally Growing AT5BI Cells do not Exhibit Mitosis-associated Increases in γ-H2AX

  Panel A is a representative DIM image (100x) 3D projection that demonstrates the lack of a qualitative difference between mitotic (white arrow) and interphase cells immunofluorescently labeled with anti-γ-H2AX and counterstained with DAPI. The merged image presents the DAPI (red) and -γ-H2AX (green) channels. Scale bar represents 2μm. Panel B presents a single representative flow cytometric dot plot of an asynchronous AT5BI population immunofluorescently labeled with anti-γ-H2AX and counterstained with PI. The dot plot depicts γ-H2AX (FL1-H, γ-axis) signal intensity versus PI (FL2-A, x-axis) signal intensity. The inset represents the PI-only pattern and demonstrates that the cells were cycling. Panel C is a representative immunoblot comparing the abundance of γ-H2AX in an asynchronous population (A) and a mitotically-enriched (M) population. Acid-solublized, nuclear protein preparations from AT5BI cells were resolved on 15% SDS PAGE gels, and immunostained with anti-γ-H2AX. The CPTS stained PVDF membrane has been included and serves as a loading control.

• Supplemental Figure 8 - Comparitive Localization of γ-H2AX and 53BP1.

  An image series of an Indian muntjac fibroblast stained for γ-H2AX (green) and 53BP1 (red) were deconvolved and then maximum point intensity (top row) or surface (bottom row) projections were generated from the data sets. The arrows indicate examples of foci where there is significant enrichment of both proteins. The boxes enclose larger foci that are enriched in 53BP1 but not γ-H2AX .

• Supplemental Tables 1 and 2