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. 2012 Sep 1;23(17):3450–3460. doi: 10.1091/mbc.E11-12-1009

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© 2012 Velichko et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 2: — Patterns of HS-induced γH2AX foci are dependent on the cell cycle phase. (A) The percentage of HS-treated (45.5°C, 30 min) mcf-7 cells possessing different numbers of γH2AX foci. More than 200 individual cells immunostained with anti-γH2AX antibody were analyzed. (B) Flow cytofluorometric analysis of the cell cycle distributions of the control (untreated) and heat-stressed cells. Aliquots of asynchronously proliferating control mcf-7 cells and cells that were heat-shocked for 30 min at 45.5°C were stained with propidium iodide and analyzed by flow cytometry. The relative DNA content of 500,000 cells was plotted against the cell number in each histogram. Calculated percentages of S-phase cells in each case are indicated below the corresponding histograms. (C) Human mcf-7 cells were pulse-labeled with BrdU (100 μM, 20 min), heat-stressed (45.5°C, 30 min), and double-immunostained against γH2AX and BrdU. The DNA was stained with DAPI. A superimposition of the green and red channels is shown in the last section (Merge). Scale bar: 20 μm. (D) Human mcf-7 cells were pulse-labeled with EdU (10 μM, 30 min), heat-stressed (45.5°C, 30 min), and immunostained against γH2AX and cyclin B1. EdU was revealed by Click Chemistry. A superimposition of the green, red, and blue channels is shown in the last section (Merge). Scale bar: 20 μm.