Lab Meeting

1. 在TW01TetER及TW05TetER上Serum starvation的條件: 6-well種50000-100000 incubate 48h，serum free組與complete medium組相比，serum free組細胞數少，細胞長得細細長長，在細胞數與細胞型態上有差異性。 補充：根據之前flow data (call cycle analysis) 看48小時的實驗，細胞6-well種100000不至於過滿，200000細胞會過滿。所以我偏愛100000 incubate 48h的condition。接著模仿MB教授的實驗方法看看serum (glucose順道一起看)對Rta-induced cellular senescence.的重要性。(Ref） 2. Establishment of TW05-EREV： 結果： (1) PEG濃縮病毒可以提高virus infection的效率（5%至20%, combine TGFbeta可達到30%），而TGFbeta則效果不彰。 (2) 細胞先放大至6-cm dish，再種1000顆細胞至10 cm dish進行G418 selection的過程中細胞便死光。 討論： 從12-well放大不要一下就放到6-cm dish中，建議先至6-well較適當。而G418 selection的步驟應該要在細胞放大時就做，防止沒有病毒的細胞勢力擴大。 若現在手邊的細胞已經沒有綠光的存在了，便重新開始以濃縮病毒的方式感染細胞。

1. The long-term kinetics (0-6 days) of Rta in Dox-treated TW01TetER The expression of p53, p21, SFN and p27 were increased in Rta induction. Increasing phosphorylation of H2AX was observed on day5 and day6 after Rta induction.2. Compare the expression level of p21, SFN, c-Myc, p53 and p63 in Dox-treated TW01TetER and TW05TetERThe expression of

1. The long-term kinetics (0-6 days) of Rta in Dox-treated TW01TetER2. The cytotoxicity of U0126, Rapamycin and LY294002 on TW01TetER3. Effect of serum starvation on the cell morphology, cell number and cellular hypertrophy

報告：1. The expression kinetics (0-72 h) of cell cycle regulators and viral immediate-early proteins in Dox-treated TW01EREV-2716. (Rta, EAD, p53, SFN)2. The cytotoxicity of U0126 on TW01EREV-2716: 72hr, IC50=45.76uM3. Pretreat 20uM U0126 1hr before dox treatment can inhibit activation of MAPK (ERK 1/2), but don’t inhibit EBV reaction in 2716.討論：首先先釐清1. Cell density與cell growth/senescence/death以及 viral reactivation有關2.

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

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。

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)

1. To compare expression levels of various host proteins in TW01-EREV#27 with its parental cell TW01TetER#16.2. Test p53 and CDK6 antibodies from ICON.