Figure 3.
Figure 3. CSK+R extraction allows super-resolution imaging of DNA damage sensors and chromatin marks at DNA ends. (A) U2OS cells were treated with IR, postincubated for 5 min, preextracted with CSK+R, and processed for immunofluorescence. Cells were analyzed by high-resolution (top left, conventional) or SIM (top right, 3D-SIM). At the bottom, images are magnifications of the boxed regions highlighted by arrowheads in the top images, highlighting the resolution gain between high-resolution (bottom left) and super-resolution (bottom right) microscopy. (B) Graph showing the fluorescence profile, in arbitrary units (AU), of an individual 3D-SIM Ku focus, corresponding to the dashed line in the bottom right panel of A. The data shown are representative of 30 measured foci in two independent experiments. (C) Frequency distribution of the number of Ku foci per γ-H2AX focus. U2OS cells were treated with 2 Gy of IR, postincubated for 5 min, preextracted with CSK+R, and processed for immunofluorescence. 3D-SIM pictures were acquired and manually analyzed for Ku foci, as represented by a frequency distribution. The data shown are from a single representative experiment out of two repeats. For the experiment shown, n = 535. (D) Ku and γ-H2AX spatial distributions as analyzed by 3D-SIM. Representative 3D-SIM pictures analyzed in C are presented. (E) U2OS cells were untreated (NT) or treated with 10 Gy of IR, postincubated for 5 min, and analyzed by immunofluorescence for NBS1 or γ-H2AX, without CSK preextraction (no CSK) or with CSK+R preextraction. (F) U2OS cells were untreated (NT) or treated with 10 Gy of IR, postincubated for 5 min, preextracted with CSK+R, and processed for immunofluorescence. Insets represent initial and 3.3-fold magnifications from boxed regions. (G) U2OS cells were treated with 2 Gy of IR, postincubated for 5 min, preextracted with CSK+R, and processed for immunofluorescence. 3D-SIM pictures were acquired, and representative foci are presented. The position of each nucleus, as defined by DAPI staining, is highlighted by a dotted line. Bars: (white) 10 µm; (green) 1 µm; (red) 0.2 µm.

CSK+R extraction allows super-resolution imaging of DNA damage sensors and chromatin marks at DNA ends. (A) U2OS cells were treated with IR, postincubated for 5 min, preextracted with CSK+R, and processed for immunofluorescence. Cells were analyzed by high-resolution (top left, conventional) or SIM (top right, 3D-SIM). At the bottom, images are magnifications of the boxed regions highlighted by arrowheads in the top images, highlighting the resolution gain between high-resolution (bottom left) and super-resolution (bottom right) microscopy. (B) Graph showing the fluorescence profile, in arbitrary units (AU), of an individual 3D-SIM Ku focus, corresponding to the dashed line in the bottom right panel of A. The data shown are representative of 30 measured foci in two independent experiments. (C) Frequency distribution of the number of Ku foci per γ-H2AX focus. U2OS cells were treated with 2 Gy of IR, postincubated for 5 min, preextracted with CSK+R, and processed for immunofluorescence. 3D-SIM pictures were acquired and manually analyzed for Ku foci, as represented by a frequency distribution. The data shown are from a single representative experiment out of two repeats. For the experiment shown, n = 535. (D) Ku and γ-H2AX spatial distributions as analyzed by 3D-SIM. Representative 3D-SIM pictures analyzed in C are presented. (E) U2OS cells were untreated (NT) or treated with 10 Gy of IR, postincubated for 5 min, and analyzed by immunofluorescence for NBS1 or γ-H2AX, without CSK preextraction (no CSK) or with CSK+R preextraction. (F) U2OS cells were untreated (NT) or treated with 10 Gy of IR, postincubated for 5 min, preextracted with CSK+R, and processed for immunofluorescence. Insets represent initial and 3.3-fold magnifications from boxed regions. (G) U2OS cells were treated with 2 Gy of IR, postincubated for 5 min, preextracted with CSK+R, and processed for immunofluorescence. 3D-SIM pictures were acquired, and representative foci are presented. The position of each nucleus, as defined by DAPI staining, is highlighted by a dotted line. Bars: (white) 10 µm; (green) 1 µm; (red) 0.2 µm.

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