XB-IMG-126411
Xenbase Image ID: 126411
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Figure 4. DSB resection occurs in cycling mitotic Xenopus extract and mitosis of human cells. (A) Schematic timeline of the cycling mitotic extract experiment. Cycling extract was incubated with sperm chromatin (5,000 sperm/µl). Nocodazole was added at 96 min to trap the nuclei in the subsequent mitosis. Microscopy analysis 34 min later confirmed that the chromatin was in a highly condensed state indicative of mitosis. An aliquot was taken before addition of 0.05 U/µl PflMI restriction endonuclease at 163 min (time 0). Aliquots were also taken at 10, 30, and 60 min after addition of PflMI and processed for chromatin isolation and Western blotting. (B) Resection of DSBs occurs in nocodazole-arrested mitotic extract. After arrest with nocodazole, DSBs were induced in the mitotic chromatin, and aliquots were taken before and at the indicated time points (minutes) after addition of PflMI and processed for chromatin isolation and Western blotting with the indicated antibodies. The mitotic status of the extract was also confirmed by the presence of pSer10–histone H3. (C) Resection of DSBs occurs in mitosis of human cells. Asynchronous HeLa cells were grown on 8-well chamber slides and mock irradiated (no damage) or microirradiated using a high-energy UV laser microscope (PALM MicroBeam IV). After the indicated time (±4 min), slides were processed and stained with antibodies against human RPA34 and phospho–Ser10-histone H3. Bar, 10 µm. Image published in: Peterson SE et al. (2011) © 2011 Peterson et al. Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View |