1、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 M) for 1 h. Cells were subsequently washed with PBS and incubated for 18 h in fresh medium supplemented with nocodazole to trap cells
2、 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 add
3、ition 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 t
4、o enter mitosis by caffeine addition (recovery),doxorubicin：Molecular Weight: 579.98mp ：216 C (dec.)(lit.) solubility：H2O: 10 mg/mL, clear, red-orange DMSO: soluble H2O: soluble ethanol: soluble methanol: soluble tetrahydrofuran: soluble storage temp. 2-8CCaffeine：Molecular Weight: 194.19mp ：233-238
5、 C 234-236.5 C(lit.)Solubility：H2O: 15 mg/mLThymidine：Molecular Weight:242.23Soluble in waterNocodazole：Molecular Weight: 301.32form ：powder color ：white mp ：300 C (dec.) solubility： DMSO: 10 mg/mL, soluble H2O: insolubleHuman U2OS osteosarcoma cells, 293T human embryonic kidney cellsA： TT 处理 18h，释放
6、 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 0.5M 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 (1M (300nM, nm2004, 10, 262-267) + BI2536 (300 nM (100
7、 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 nature 的详细方法B：TT 处理 18h，释放 9h，处理 18h，释放 7h，Hela 进入 G2 期；PBS 洗后（不要用 PBS，请用无血清培养基 ）用 100ng/ml nocodazole+VX680 (1M)+BI2536(300 nM) （
8、VX680 和BI2536 包括单独处理以及联合处理）处理培养 1h，3h，5h，7h ；检测通过 anti-pS10-H3 染色 ，流式测定 M 期细胞比例。问：以后是否需要做 VX680 和 BI2536 浓度的梯度？第一次实验：7：00a.m. 换新鲜培养基释放14：00p.m. 进入 G2phase，0.5M doxorubicin 处理 1h15：00p.m. 洗掉后，第一个六孔板第一行加 nocodazole (100ng/ml)+ Caffeine (5mM) + BI2536(100 nM).二个板其他孔洗掉后，加 nocodazole (100ng/ml)+ Caffeine
9、 (5mM)，所有孔拍照。18：00p.m. 第一个板第二行加 BI2536(100 nM). 所有孔拍照。21：00p.m. 第二个板第一行加 BI2536(100 nM). 所有孔拍照。24：00p.m. 所有孔拍照。第二次实验：9:00a.m. Thymidine 洗掉，释放 7h16:00p.m. Doxorubicin 处理 A,B,C,D 组 1h17:00p.m. 全部孔加药,并拍照20:00p.m. 全部孔拍照23:00p.m. 全部孔拍照2:00a.m. 全部孔拍照5:00a.m. 全部孔拍照8:00a.m. 全部孔拍照11:00a.m. 全部孔拍照DNA damage:A:
10、noc+caf+vxB:noc+caf+BIC: noc+caf+vx+BID: noc+cafNormal:E: noc+VXF: noc+BIG: noc+ vx+BIH: nocThymidine :2 mMDoxorubicin: 0.5MNocodazole: 100ng/mlVX-680:300nMBI2536:100nM 第三次实验（FACS&Western Blot）：每个组要 6 孔一组一个板：1st Day 4:00a.m. Thymidine 处理 21h2nd Day 1:00a.m. Thymidine 洗掉，释放 7h8:00a.m. Doxorubicin 处理
11、A,B,C,D 组 1h9:00a.m. 所有组加药，每个组收一孔3:00p.m. 每个组收一孔5:00p.m. 每个组收一孔7:00p.m. 每个组收一孔9:00p.m. 每个组收一孔11:00p.m. 每个组收一孔配 sample buffer，ripa.生理盐水（PBS）DNA damage:A:noc+caf+vxB:noc+caf+BIC: noc+caf+vx+BID: noc+cafNormal:E: noc+VXF: noc+BIG: noc+ vx+BIH: nocThymidine :2 mMDoxorubicin: 0.5MNocodazole: 100ng/mlVX-680:300nMBI2536:100nM
