1. to observe whether CTCF interact with EBV Rta by Immunoprecipitation assay
a) in TW01 inducible cell lines, CTCF and Flag-Rta can not be immunoprecipitated by each other. I also tried Flag beads, which are provided by Sigma, both of Flag-Luc and Flag-ER caught CTCF. I can not prove the interaction between CTCF and EBV Rta in TW01 inducible cell lines.
b) in co-transfected HEK293/293FT cell lines(Flag-Luc/ER/KR), CTCF can immunoprecipitate Flag-ER. However, the ratio of each plasmid is unfair, i will adjust the amount of these plasmids (may ignore Luc) and perform this experiment again.
2. to screen 12 putative target genes in CTCF/Flag-ChIP samples
利用Q-PCR的方式, 這12個genes無法在CTCF-ChIP的samples中被偵測到; 但利用一般PCR的方式, 則可偵測到JUN及H19(雖然band很微弱)
a) 在ChIP之前的sonication片段可能太短, 以至於所設計的這些將近500 bp PCR product的primer不能bind上DNA
b) ChIP的效率不好, 在抗體/磁珠/sample量的比例上該做些調整
Aug 31, 2011
Aug 29, 2011
8/24 Lab Meeting-ingrid
(1) 用IP將KR抓下，用WB(RL2)來確認O-GlcNAc變化程度；經更改IP流程，可以有效率以KR(660) from ICON將KR抓下(ERKV_Dox48h)，但是之後應用於ERKV_shLuc_Dox48h、ERKV_shOGTA1_Dox48h及ERKV_shMGEA5B2_Dox48h，卻效率不佳，以致於後來的RL2及PARP1皆無法辨認。目前持續改進IP流程中。Note:如果是要看KR蛋白質，建議Lysis buffer要加phosphotase inhibitor以避免KR的碎裂。
(2) 關於進行shRNA的Q-PCR，已完成實驗數據。但是分析時，應注意星號的標誌，正確為*p<0.05, **p<0.01, ***p<0.001(星號應水平標誌)。
(3) 經由計算細胞生長速率及考慮表現Luc蛋白質的程度，混合細胞得到TW01TetLuc pool :#2+ #8及TW05TetLuc pool:#1+ #3+ #5。
(4) 三種Flag Ab測試(Sigma F1804舊、Cell Signaling及Sigma F1804新)：認識Luc蛋白質在TW01TetLuc pool及TW05TetLuc pool的titer皆差不多，除了Cell Signaling背景比較髒。
(2) 關於進行shRNA的Q-PCR，已完成實驗數據。但是分析時，應注意星號的標誌，正確為*p<0.05, **p<0.01, ***p<0.001(星號應水平標誌)。
(3) 經由計算細胞生長速率及考慮表現Luc蛋白質的程度，混合細胞得到TW01TetLuc pool :#2+ #8及TW05TetLuc pool:#1+ #3+ #5。
(4) 三種Flag Ab測試(Sigma F1804舊、Cell Signaling及Sigma F1804新)：認識Luc蛋白質在TW01TetLuc pool及TW05TetLuc pool的titer皆差不多，除了Cell Signaling背景比較髒。
Aug 23, 2011
8/17 Lab meeting-蔡小丸
1. 在HEK293細胞中共同表達KR, S634A/S636A, Luc及不同量的Pin1, 結果發現大量的Pin1會造成KR, S634A/S636A, Luc蛋白量的降低, 由於Luc已知不會與Pin1交互作用, 因此大量Pin1可能部份造成細胞general的影響了。
2. IP結果初步看到, KR及S634A/S636A會被sumo-1所修飾, Luc則沒有, S634A/S636A被sumo-1修飾的能力較差, 但仍需進一步實驗確認sumo-1修飾KR及S634A/S636A所扮演的角色。
2. IP結果初步看到, KR及S634A/S636A會被sumo-1所修飾, Luc則沒有, S634A/S636A被sumo-1修飾的能力較差, 但仍需進一步實驗確認sumo-1修飾KR及S634A/S636A所扮演的角色。
Aug 11, 2011
8/10 Lab meeting
1. The trial of new medium KGM-2 ── comparing with medium DKSFM/EpiLife for culturing NP460hTert:
(1) Cells culturing in KGM-2 showed more like triangle shape rather than elongated morphology as seeding in DKSFM/EpiLife.
(2) Cells culturing in KGM-2 had slower proliferation rate (Doubling time:55.4hrs) than culturing in DKSFM/EpiLife medium (Doubling time:36hrs).
2.Different [Ca2+] treatments for 6 days in KGM-2 medium for NP460hTert
(1) Along the treatment, cells in 2 calcium free treatments showed poor adhesion to the dish (細胞沒有延伸或突觸等的貼附形態出現). *Mortality rate:32% and 35%.
(2) Cells in 0.1mM [Ca2+] showed good adhesion (細胞亦無層疊的現象) and had the lowest mortality rate (11%) among all treaments.
(3) Cells in 0.5mM,1mM and 1.5 mM Ca2+ showed the tendency to cluster and pile up together under high Ca2+ culture.(第四天後細胞堆疊明顯)
(4) Cells in 1.5mM Ca2+ showed the highest mortality rate (48%), almost 50% of the cells were dead at the measuring time. The mortality of 1mM Ca2+ treatment weas 24%, 0.5mM Ca2+ was27%.
(5) After trypisinization, cells didn't look bigger when counting.(通常要準備走上senescence或分化的細胞會變較大)
討論結果:
(1) KGM-2和DKSFM/EpiLife下細胞生長形態的照片因為seeding不均勻的關係，要再重新試驗新種下比較均勻的10cm dish中再照一次。(8/10已重照完畢)
(2) 不同鈣離子濃度的試驗因為細胞種的不均勻可再做一次試驗(等下禮拜一母盤有夠多的細胞)，而其中一個無鈣離子的對照組可改為0.05mM來試驗(0.1mM still a little bit high or not?!)
(3) 下禮拜可以使用不同濃度的retinoic acid treatment!(in medium DKSFM/EpiLife or KGM-2)，可試試看曹老師用serum來促進differentiation的方法!
Aug 10, 2011
08/10 Lab Meeting- 小夏
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。
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。
NP460hTERT in KGM2
由 sufang 在 三, 08/10/2011 - 14:54 發表 Older Posts EBV infection EBV Rta NP460hTERT transient lytic
2011/08/10 Lab Meeting
1. The trial of new medium KGM-2 ── comparing with medium DKSFM/EpiLife for culturing NP460hTert:
(1) Cells culturing in KGM-2 showed more like triangle shape rather than elongated morphology as seeding in DKSFM/EpiLife.
(2) Cells culturing in KGM-2 had slower proliferation rate (Doubling time:55.4hrs) than culturing in DKSFM/EpiLife medium (Doubling time:36hrs).
2.Different [Ca2+] treatments for 6 days in KGM-2 medium for NP460hTert
(1) Along the treatment, cells in 2 calcium free treatments showed poor adhesion to the dish (細胞沒有延伸或突觸等的貼附形態出現). *Mortality rate:32% and 35%.
(2) Cells in 0.1mM [Ca2+] showed good adhesion (細胞亦無層疊的現象) and had the lowest mortality rate (11%) among all treaments.
(3) Cells in 0.5mM,1mM and 1.5 mM Ca2+ showed the tendency to cluster and pile up together under high Ca2+ culture.(第四天後細胞堆疊明顯)
(4) Cells in 1.5mM Ca2+ showed the highest mortality rate (48%), almost 50% of the cells were dead at the measuring time. The mortality of 1mM Ca2+ treatment weas 24%, 0.5mM Ca2+ was27%.
(5) After trypisinization, cells didn't look bigger when counting.(通常要準備走上senescence或分化的細胞會變較大)
討論結果:
(1) KGM-2和DKSFM/EpiLife下細胞生長形態的照片因為seeding不均勻的關係，要再重新試驗新種下比較均勻的10cm dish中再照一次。(8/10已重照完畢)
(2) 不同鈣離子濃度的試驗因為細胞種的不均勻可再做一次試驗(等下禮拜一母盤有夠多的細胞)，而其中一個無鈣離子的對照組可改為0.05mM來試驗(0.1mM still a little bit high or not?!)
(3) 下禮拜可以使用不同濃度的retinoic acid treatment!(in medium DKSFM/EpiLife or KGM-2)，可試試看曹老師用serum來促進differentiation的方法!
2 comments:
SFL 8/11/11, 11:21 AM
Yes,as Ingrid pointed out the "old" medium contains Ca2+ at a concentration of (60uM+<100um 0.1mm="" 30="" 50um="" a="" about="" after="" although="" and="" but="" can="" cells="" change="" chronic="" cut="" difference.="" does="" down="" far="" give="" good="" gradually="" if="" ingrid="" it="" make="" may="" natures="" p="" s="" seems="" so="" some="" that="" their="" to="" try.="" um="" we="" while="" why="" worried="" xxum="">
Yes,as Ingrid pointed out the "old" medium contains Ca2+ at a concentration of (60uM+<100um 0.1mm="" 30="" 50um="" a="" about="" after="" although="" and="" but="" can="" cells="" change="" chronic="" cut="" difference.="" does="" down="" far="" give="" good="" gradually="" if="" ingrid="" it="" make="" may="" natures="" p="" s="" seems="" so="" some="" that="" their="" to="" try.="" um="" we="" while="" why="" worried="" xxum="">
小嬿 8/11/11, 12:50 PM
仲峻學長看過母盤NP460的照片覺得細胞的morphology跟他之前的印象不一樣(現在的細胞比剛recover時更長, 且有些交疊),會不會我們現在用的DKSFM+EpiLife不夠新鮮,導致他變形?(手上medium已經快用完了,將準備新的1L medium~)
仲峻學長看過母盤NP460的照片覺得細胞的morphology跟他之前的印象不一樣(現在的細胞比剛recover時更長, 且有些交疊),會不會我們現在用的DKSFM+EpiLife不夠新鮮,導致他變形?(手上medium已經快用完了,將準備新的1L medium~)
08/10/2011
Asia/Taipei
Aug 4, 2011
08/03 Lab Meeting-小嬿
進度報告
1. 在HEK293分別做Flag-Luc/ER/KR/gZ的transient transfection 24hrs,
a. IP CTCF: 1/6 of IP中, WB CTCF未有band-->用5/6的memebrane check again;
5/6 of IP中看到ER (by 467), 微弱的gZ(by 4F10), 未見KR(by Yoshi's);
b. IP Flag: 失敗! 1/20 of IP可見Flag蛋白被IP下來, 但未見CTCF!
@WHT建議二抗濃度可再做調整, 讓background更乾淨(e.g. ~100 kD的band)
@在TW01系統試試看!
@SFL建議在293FT做transfection效果更佳! (plus co-co-co-transfection!)
2. 利用ChIP分析TW01TetER_16細胞中, 在Dox induction的狀況下, CTCF所binding的DNA fragment量是否有所改變
-->依據Q-PCR結果顯示, input DNA量不穩定, 影響CTCF IP的數值計算; mock(IgG) IP所得DNA量較少, Ct值SD非常大
@SFL建議做少一點sample量, IP CTCF和Rta, 並且screen先前擬定偵測的gene
GOGOGO! 往PLoS Pathogen邁進!!!
1. 在HEK293分別做Flag-Luc/ER/KR/gZ的transient transfection 24hrs,
a. IP CTCF: 1/6 of IP中, WB CTCF未有band-->用5/6的memebrane check again;
5/6 of IP中看到ER (by 467), 微弱的gZ(by 4F10), 未見KR(by Yoshi's);
b. IP Flag: 失敗! 1/20 of IP可見Flag蛋白被IP下來, 但未見CTCF!
@WHT建議二抗濃度可再做調整, 讓background更乾淨(e.g. ~100 kD的band)
@在TW01系統試試看!
@SFL建議在293FT做transfection效果更佳! (plus co-co-co-transfection!)
2. 利用ChIP分析TW01TetER_16細胞中, 在Dox induction的狀況下, CTCF所binding的DNA fragment量是否有所改變
-->依據Q-PCR結果顯示, input DNA量不穩定, 影響CTCF IP的數值計算; mock(IgG) IP所得DNA量較少, Ct值SD非常大
@SFL建議做少一點sample量, IP CTCF和Rta, 並且screen先前擬定偵測的gene
GOGOGO! 往PLoS Pathogen邁進!!!
Jul 29, 2011
Jul 22, 2011
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
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
Jul 20, 2011
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?
(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?
Jul 7, 2011
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
(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.
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。
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。
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