JCS089847 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Mobility of chromatin domains containing DSBs induced by Topoisomerase inhibitor etoposide (in U2OS cells) and by γ-radiation (in normal human fibroblasts). (A) 100 randomly selected trajectories, depicting movement of the ionizing irradiation and etoposide induced DSBs in U2OS cells were plotted as originating from the same point on the 2D plane. The color of the segments of the trajectories represents time after the start of imaging. (B) MSD of the indicated types of chromatin domains. Bar graph shows MSD of the respective domains averaged over last 3 time points. Asterisks indicate statistical significance with p<0.05, assessed using the student t-test. Error bars represent SEM. At least 30 cells and at least 150 foci were analyzed per data series. (C) MSD of the indicated types of chromatin domains in normal human fibroblasts. Error bars represent SEM. At least 10 cells and at least 5 foci per cell were analyzed per data series.

• Supplemental Figure S2 -

  Fig. S2. Mobility of Cy3CDs in U2OS cells is not altered by irradiation or curcumin treatment. MSD of Cy3CDs in cells exposed to 0 or 10 Gy of γ-radiation or to 1 µM curcumin. Error bars represent SEM. At least 20 cells and at least 5 foci per cell were analyzed per data series.

• Supplemental Figure S3 -

  Fig. S3. γ-H2AX IRIF frequencies and cell cycle distribution in cells treated with TSA, curcumin and 5-azacitidine. (A) U2OS cells expressing 53BP1-GFP were incubated in the presence of the indicated inhibitor, exposed to 1Gy of γ-radiation, fixed 1, 5 or 24 h after irradiation and stained using antibodies against histone γ-H2AX. Bars indicate average number of γ-H2AX IRIF per cell. At least 100 cells were scored per data point. Error bars indicate standard deviation. (B) U2OS cells expressing 53BP1-GFP were incubated in the presence of the indicated inhibitors, as described in the supplementary materials and methods section, then incubated in the presence of BrdU and fixed. Standard cell cycle distribution analysis was then performed by FACS analysis of cells stained using antibodies against BrdU. Graph represents average percentage of cells in the indicated cell cycle phase, obtained from duplicate samples per data point. Error bars indicate range of values obtained from 2 duplicates. (C) TSA treatment increases histone acetylation. Detection of acetylated histone H3 K9 (upper panel) in cells incubated in standard medium (left lane) or in medium supplemented with 150 nM TSA for 24 h (right lane). Sample loading is controlled by probing for actin (lower panel). Panels show a representative experiment.