生技醫藥國家型計畫NRPB誠徵生力軍

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0720 Lab meeting-蔡小丸

1. 在293FT細胞中共同表達K-RTA, S634A/S636A, Luc (control protein)及Pin1, 結果發現Pin1降解K-RTA及S634A/S636A的能力相似
2. 用一批新抽的K-RTA及S634A/S636A transfection至293FT後發現, K-RTA與S634A/S636A穩定性接近, 推測634/636位點磷酸化與否與其穩定性無關
3. 完成oral cancer tissue microarray (BC34011, HN811)之DDR1 IHC染色, 未來將進行DDR1表達量之給分
4. 整理DDR1之ligans-collagens(type1-5)在一些oral cancer tissues中觀察到他們的RNA level有增加的情形, 最後我們認為在口腔癌中, collagen type1及type4為可能較重要的DDR1 ligands

7/13 Lab Meeting-ingrid

(1) 用IP將KR抓下，用WB(RL2)來確認O-GlcNAc變化程度；結果是可以成功以KR(660) from ICON將KR抓下，但是效率不佳，以致於後來的RL2及PARP1皆無法辨認。接下來會以重收細胞(以EBC Buffer溶解細胞)、增加cell lysate或KR(660)的量來改進IP效率。NOTE：以6% SDS-PAGE分析；KR(Ueda)會認到2條band?
(2) 進行Q-PCR檢查送入shOGT的293FT細胞樣本中，MGEA5的mRNA是否有減少，反之亦然。結果發現ACTIN primer會影響，但GAPDH不會影響。經重複實驗，排除off-target作用。
(3) TW01TetLuc有4株clones及TW05TetLuc有4株clones進行WB (Flag Ab) 確認為positive。接著進行計數細胞，將生長速率差不多的細胞混合。NOTE：Flag Ab是否有問題?

EBV Rta-induce cellular senescence in nasopharyngeal cells

小嬿:

(A) 搞定EBV Rta 對 CTCF 在 genome organization 之影響。

(B) 行有餘力 (and a hobby!): Establishment of TW05-EREV

Jean:

(A) Find out the best differentiation protocol for NP460hTert.

(1) reagents: TGFb, calcium …

(2) markers: p63, MYC…

(3) controls: TW05TetER and TW01TetER #16 cells

(4) Ref: 第一篇，第二篇 , 第三篇…

(B) 行有餘力: Cloning of TW05-EREV (由小嬿、小夏指導)
小夏:
Continue to figure out the effects of Rta in serum or glucose -deprived TW01 and TW05 cells.

07/06 Lab Meeting- 小夏

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 TW05TetER
The expression of p53, p21, and SFN were increased in Rta induction while the levels of c-Myc and p63 were decreased.
3. The cytotoxicity of U0126, Rapamycin and LY294002 on TW01TetER
IC50分別為：U0126 40uM, Rapamycin >10uM, LY294002 60uM
4. Test the effect of serum starvation and no glucose on the cell morphology in TW01TetER and TW05TetER
A. 在50000顆細胞的seeding下，serum starvation 48小時可以觀察到細胞生長停滯(也許是緩慢)。
B. 19號長得比16號快 (48小時內活細胞數會較16號多)，但19號也較16號快死(16號活細胞數較19號多)，我覺得19號代謝較快。
C. 不管是TW01或是TW05，細胞缺少葡萄糖最後會死亡(在第五天觀察)。而在缺乏葡萄糖48小時的情況下再添加葡萄糖回去，細胞可以回復正常生長。
D. Serum與Glucose對細胞的生長都很重要。

RNA-Seq Data USB 1 and 2

2011/07/06_
由 sufang 在 三, 07/06/2011 - 00:00 發表 Older Posts BrCa Dan Robinson RNA-seq USB

2011-07-06

2012-04-09