請大家操作實驗時，要著實驗衣，違者口頭警告三次，之後每次抓到罰鍰NTD 100。
Apr 24, 2011
Apr 15, 2011
04/13 Lab Meeting- 小夏
To test the effect of U0126 on EBV Rta-induced EBV reactivation on TW01EREV-2716
1. 初步結果72hr 20uM U0126死了一半的細胞 (WST-1 assay)
2. 感覺Dox 48小時活細胞有隨著U0126濃度的增加而增加
3. Dox 72小時細胞全部懸浮於medium，感覺72小時Dox作用效果遠大於U0126。(這樣可以說U0126是擋不住ER的Function? 也許可以像學姊早上說的降低Dox的量)
Next work will
1. Determine the cytotoxicity of U0126 on TW01EREV-2716 by WST-1 assay.
2. Confirm the expression kinetics (0-72hrs) of cell cycle regulators and viral proteins in Dox-treated TW01EREV-2716. (ex. SFN,p53)
1. 初步結果72hr 20uM U0126死了一半的細胞 (WST-1 assay)
2. 感覺Dox 48小時活細胞有隨著U0126濃度的增加而增加
3. Dox 72小時細胞全部懸浮於medium，感覺72小時Dox作用效果遠大於U0126。(這樣可以說U0126是擋不住ER的Function? 也許可以像學姊早上說的降低Dox的量)
Next work will
1. Determine the cytotoxicity of U0126 on TW01EREV-2716 by WST-1 assay.
2. Confirm the expression kinetics (0-72hrs) of cell cycle regulators and viral proteins in Dox-treated TW01EREV-2716. (ex. SFN,p53)
Apr 6, 2011
04/06 Lab Meeting 小嬿
1. 降低細胞數至7.5 x 10^6/ml做sonication的效果仍未達百分百, 將試5 x 10^6/ml的效果
2. 比較TW01TetER(19) Ctrl-24hrs及Dox-24hrs, (1) CTCF bind在H19及SFN promoter的能力似乎相當, 一般PCR及Q-PCR所看到的差距不大 (2) 初步測試Rta也可bind在H19(weak)及SFN promoter上, Q-PCR 確認中
Apr 5, 2011
0330 Lab meeting-蔡小丸
1. 利用coexpress Flag-K-RTA及Flag-CDK9的lysates 進行CDK9 IP實驗 結果發現K-RTA, S634A/S636A, NLSm均與CDK9有interaction, K-RTA與CDK9的association較S634A/S636A強 2. Pin1 isomerase mutant K34A與K-RTA大量共同表達 K-RTA的molecular weight並沒有改變, 初步推測 Pin1的isomerase的活性並不會影響K-RTA的molecular weight
Mar 31, 2011
3/30 Lab Meeting-ingrid
(1) 以shLuc、shMGEA5的lentivirus (稀釋倍數1/8) 送入ERKV (2.4x10^5/ml/well/12well) 中，24、48、72小時後，收lysates進行WB確認目標蛋白質的確有減少，B2效果比D2好。shLuc的lysates中，MGEA5的表現隨著時間增加而增加(?)。
(2) 以shLuc、shMGEA5的lentivirus (稀釋倍數1/8) 送入ERKV (2.4x10^5/ml/well/12well) 中，48小時後加Dox，再48小時收lysates進行WB確認，MGEA5減少最多的B2，所引發的lytic reactivation越強烈。加入Dox也會引起MGEA5表現增加(?)。總之，將D2提高至1/4，再加入2個NC(有shRNA但未加Dox)，重做一次。
(2) 以shLuc、shMGEA5的lentivirus (稀釋倍數1/8) 送入ERKV (2.4x10^5/ml/well/12well) 中，48小時後加Dox，再48小時收lysates進行WB確認，MGEA5減少最多的B2，所引發的lytic reactivation越強烈。加入Dox也會引起MGEA5表現增加(?)。總之，將D2提高至1/4，再加入2個NC(有shRNA但未加Dox)，重做一次。
Mar 24, 2011
03/24 Lab Meeting- 小夏
1. U0126 (MEK1/2 inhibitor) can inhibit EBV Rta induced p21 expression and EBV reactivation in EREV8.
2. caspase 3 is involved in the survival activity of DDR1 in OSCC (在OEC-M1 and TW2.6 中knockdown DDR1看到pro form的減少)。
3. 用autophage marker LC3B看看autophage是否參予著DDR1調控生長的機制。
2. caspase 3 is involved in the survival activity of DDR1 in OSCC (在OEC-M1 and TW2.6 中knockdown DDR1看到pro form的減少)。
3. 用autophage marker LC3B看看autophage是否參予著DDR1調控生長的機制。
Mar 23, 2011
03/16 Lab Meeting-小嬿
To test Magnetic ChIP method:
1. non-specific band was less than agarose method
2. By using standard end-point PCR analysis, RNA Pol II is able to bind promoters of GAPDH, H19, and SFN; CTCF can bind H19 promoter and weakly on SFN promoter.
3. The problem of sonication efficiency is not dissolved yet.
1. non-specific band was less than agarose method
2. By using standard end-point PCR analysis, RNA Pol II is able to bind promoters of GAPDH, H19, and SFN; CTCF can bind H19 promoter and weakly on SFN promoter.
3. The problem of sonication efficiency is not dissolved yet.
Mar 22, 2011
3/16 Lab Meeting-ingrid
(1) Dox誘導型細胞株的選殖
TW05TetLuc：#1-6測試中
TW05TetER：#2-18的WB有認到ER，重新做IFA以確認表現蛋白質的比例及強弱
TW05TetER/gZ：#1-33測試中
(2) 以SK-N-SH cell、HelaTet、TW03Tet及TW05Tet lysates做OGT Ab的western analysis，用黃老師家的goat二抗，沒有認到目標蛋白質，重買OGT Ab。
(3) 以shLuc、shMGEA5的lentivirus (稀釋倍數1/8) 送入ERKV (2.4x10^5/ml/well/12well) 中，24、48、72小時後，收lysates進行WB確認目標蛋白質的確有減少，B2效果比D2好。
TW05TetLuc：#1-6測試中
TW05TetER：#2-18的WB有認到ER，重新做IFA以確認表現蛋白質的比例及強弱
TW05TetER/gZ：#1-33測試中
(2) 以SK-N-SH cell、HelaTet、TW03Tet及TW05Tet lysates做OGT Ab的western analysis，用黃老師家的goat二抗，沒有認到目標蛋白質，重買OGT Ab。
(3) 以shLuc、shMGEA5的lentivirus (稀釋倍數1/8) 送入ERKV (2.4x10^5/ml/well/12well) 中，24、48、72小時後，收lysates進行WB確認目標蛋白質的確有減少，B2效果比D2好。
Mar 10, 2011
0309 Lab meeting-蔡小丸
1. 共同表達K-RTA與Pin1後發現, 大量Pin1表達會造成 K-RTA降解
2. 初步結果顯示 K-RTA與 Pin1間有交互作用, 但需加入IgG控制組
3. 完成silence DDR1對於口腔癌細胞株生長之影響
2. 初步結果顯示 K-RTA與 Pin1間有交互作用, 但需加入IgG控制組
3. 完成silence DDR1對於口腔癌細胞株生長之影響
Mar 7, 2011
03/02 Lab Meeting-小嬿
1. check Q-PCR primers: p21 and SFN-->Workable!
2. ChIP test :
a. antibodies: mouse IgG, CTCF and Flag antibody
b. primers: H19ICR, SFN, GAPDH(coding region)
c. results: (1) the problem of sonication is not dissolved
(2) condition of Q-PCR is not well, but standard end-point PCR is OK
(3) input is not equal. (one of them is disappeared, actually)
(4) the amount of DNA fragment bound by mouse IgG was similar to CTCF
d. Future work:
(1) to try magnetic system by using standard end-point PCR (IgG and CTCF)
(2) to test Q-PCR condition
2. ChIP test :
a. antibodies: mouse IgG, CTCF and Flag antibody
b. primers: H19ICR, SFN, GAPDH(coding region)
c. results: (1) the problem of sonication is not dissolved
(2) condition of Q-PCR is not well, but standard end-point PCR is OK
(3) input is not equal. (one of them is disappeared, actually)
(4) the amount of DNA fragment bound by mouse IgG was similar to CTCF
d. Future work:
(1) to try magnetic system by using standard end-point PCR (IgG and CTCF)
(2) to test Q-PCR condition
Mar 2, 2011
03/02 Lab Meeting- 小夏
No SFN signal in TW01TetEREV#27 cells may be due to GFP antibody-blocking. (Sorry didn’t think of it, I’ll check the probability)
2/23 Lab Meeting-ingrid
報告進度
(1) TW05TetER：#1-18中，除了#1及#12以外，其餘在IFA中，為FLAG Ab陽性，重複做IFA(點片)以重複確認。
(2) 以SK-N-SH cell lysates做OGT Ab的western analysis，覺得沒有認到目標蛋白質，再以Hela cell lysates當positive control及黃老師家的goat二抗再確認。
(1) TW05TetER：#1-18中，除了#1及#12以外，其餘在IFA中，為FLAG Ab陽性，重複做IFA(點片)以重複確認。
(2) 以SK-N-SH cell lysates做OGT Ab的western analysis，覺得沒有認到目標蛋白質，再以Hela cell lysates當positive control及黃老師家的goat二抗再確認。
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