DNA损伤实验.docxVIP

To induce DNA damage in G2, medium was first replaced to release cells from the thymidine block, and at 7 h into this release cells were treated with doxorubicin (0.5 μM) for 1 h. Cells were subsequently washed with PBS and incubated for 18 h in fresh medium supplemented with nocodazole to trap cells in mitosis. Where indicated, DNA-damage-signalling was silenced by addition of caffeine (5 mM). For reconstitution assays, expression of PLK1 and the respective mutants was induced by addition of tetracycline (1 mgml21). To determine the rate of recovery, cells were harvested 9 h after caffeine addition and fixed in ice-cold ethanol (70%). cells were synchronized at prometaphase by a thymidine nocodazole arrest (TN; a 18-hr thymidine arrest and a 5-hr release, followed by a 14-hr nocodazole arrest). Mitotic cells were then collected by shake-off. arrested in G2 with doxorubicin and stimulated to enter mitosis by caffeine addition (recovery), doxorubicin：Molecular Weight: 579.98 mp ：216 °C (dec.)(lit.) solubility： H2O: 10 mg/mL, clear, red-orange DMSO: soluble H2O: soluble ethanol: soluble methanol: soluble tetrahydrofuran: soluble storage temp. 2-8°C Caffeine：Molecular Weight: 194.19 mp ：233-238 °C 234-236.5 °C(lit.) Solubility： H2O: 15 mg/mL Thymidine： Molecular Weight: 242.23 Soluble in water Nocodazole： Molecular Weight: 301.32 form ：powder color ：white mp ：300 °C (dec.) solubility： DMSO: 10 mg/mL, soluble H2O: insoluble Human U2OS osteosarcoma cells, 293T human embryonic kidney cells A： TT 处理18h，释放9h，处理18h，释放7h，Hela进入G2期； thymidine (2.5 mM, 24 h) treatment即可（参照nature及MolCell2004-15-799文章），释放后5小时后(U2OS细胞；Hela细胞应该为多少小时？)使用0.5μM doxorubicin处理1h，PBS洗后（不要用PBS，请用无血清培养基）用nocodazole (100ng/ml (50 ng/ml))+ Caffeine (5mM) + 【VX680 (1μM (300nM, nm2004, 10, 262-267) + BI2536 (300 nM (100 nM, currbiol2007-17-304-315)】（VX680和BI2536包括不处理、单独处理以及联合处理）处理培养0h，2h，4h，6h(是在该时间点加抑制剂后于某一相同时间(9hr)收细胞，还是在起始点同时加抑制剂而于该时间点收细胞)；检测 通过anti-pS10-H3染色 ，流式测定M期细胞比例。请参加natu