Dec 21, 2009
Dec 18, 2009
新的挑戰—for YCK (更新)
真是不好意思,我又改變順序了
1. 回溶HeLaTet (#3、#4)及HONE1Tet
2. 測試Bsd在TW03、CGHNK2及Zeo對HeLaTet、HONE1Tet的耐受性
3. 293FT用來包pLentiTR6, pLentiFlag-gZ, -cZ及GFP (每種包3個well, 就是半盤6-well plate之意).
同時,可否問一下進階業務,"LONZA"有款叫KGM的medium,專門用來養keratinocyte,是否有進一部資料可供參考! 謝謝.
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on 12/2/2009 Crazy SFL wrote:
哈哈哈, 新年新氣象,這是給YCK和YLC的小禮物。先把手邊的東西忙完後,請著手下面有趣的實驗:
1. 回溶HeLaTet (#3、#4)及HONE1Tet
2. 測試Bsd在TW03、CGHNK2及Zeo對HeLaTet、HONE1Tet的耐受性
3. 293FT用來包pLentiTR6, pLentiFlag-gZ, -cZ及GFP (每種包3個well, 就是半盤6-well plate之意).
同時,可否問一下進階業務,"LONZA"有款叫KGM的medium,專門用來養keratinocyte,是否有進一部資料可供參考! 謝謝.
----------------------------------------------------------
on 12/2/2009 Crazy SFL wrote:
哈哈哈, 新年新氣象,這是給YCK和YLC的小禮物。先把手邊的東西忙完後,請著手下面有趣的實驗:
- Try to establish TW03-TetR, CGHNK2-TetR, CGHNC8-TetR and TW2.6-TetR。
- Try to establish Luc, ER and gZ (B95-8 strain) in HONE1Tet, NP460Tet, TW03Tet, CGHNK2Tet, CGHNC8Tet and TW2.6Tet。這裡有個重點就是請再重讀一下原instruction manual, 把induction量弄到和293TetER相可匹配的程度。我們需要步步為營。
Dec 1, 2009
量化西方墨漬法
以後實驗室quantitate protein band請依以下步驟進行:
- Plasmid 最好是一起抽的一批, 因實驗室LB/BHI已過使用期限,希望大家每次抽質體時盡量使用新鮮media及LB plate (1-cm厚度最佳),超過一個月以上的media及plate請自行定時清理(請不要當福壽螺到處蔓延留種,謝謝)。
- 若質體中有SV40-ori, 請以293T為host (DLR/expression etc),其餘它種質體則用293。
- Transfection (24~48 h)每次請做p-100/1 ml-lysate的份量(6x p-35/1.2-ml), protein lysate量一次到位,不要老是重複在養細胞/TF效率好像有問題/plasmid不夠...(姑且稱之為小鼻子小眼睛的實驗)
- 檢測各質體間表現量:(i) 請以wild-type為標準 (所以wild-type可能要多transfect一份),293T為host的取5-ug及10-ug, 293為host的取25-ug及50-ug protein extracts run數片小膠,確定所使用的西方墨漬條件可分出一倍和兩倍間的差異。若是過強,那就請在砍primary/ secondary antibody濃度、incubation時間、ECL強度上三件事情上做調整。(ii)依前步驟所訂出的條件run一片小膠檢查各檢體間是否差異很大,如有二倍以上差異而各步驟又沒有太大疑慮,那表示可能有protein stability/proteosome involved,值得設計其他實驗來分析之.兩倍以下差異者微調input量可繼續往下的實驗。(Plasmid, 細胞用畢的請定時清理,不要當福壽螺到處蔓延留種,謝謝)。
- 附註定量software有三款,請在Lab Protocol中找到小抄。
Novartis 石明石博士 Michael Shi 演講
- TKI 258 was a FGFR1 inhibitor, but the use of TKI 258 causes FGF23/bFGF increased.
- Melanoma trial found FGF mediated escape form anti-angiogenesis (sorxxxtinb/...) Early phase: VEGFR2 blockadge.
- Phase 3 trials: (a) FGFR3 expression/t94,14) trans-location in melanoma (b) FGFR3 mutations in bladder cancer (c) FGFR1 expression in breast cancer [這裡若有蒲教授的data就好了!!]
- AUY 922 is an inhibitor of HSP90 for GI tract tumor.
- BGT226 is is on clinical trial for NPC (與中文大學馬教授合作,明年AACR發表) 另一位Mike補充陳所長有拿藥用在oral cavity (張光裕醫師 was in charge, he found this drug induced autophagic death in a panel of oral cancer cells)
- BKM120 is a PI3K -specific inhibitor; BGT226 and BE2 232 are dual kinase inhibitors (mTOR and PI3K) 這部份還是沒有搞清楚.
- ASA 404 in NSCLC.
- LED 225 is for Smoothened/Hedgehog pathway.
- MGM 黃秀芬醫師介紹 Tissue bank/LCM slides for Chlorangiocarcinoma...
Nov 21, 2009
知識分享 - immunopreciptitation
LHC大型強子對撞器→修好了
Nov 19, 2009
Nov 18, 2009
Nov 9, 2009
Nov 1, 2009
宜蘭之旅
DASL: cDNA mediated Annealing Selection, extension and Ligation (Illumina)
expression array of 40 N/T OSCC samples from Dr.蕭.
ShSSAT: growth inhibition escape. Illumina's miRNA and methylation array for 40 N/T OSCC samples from Dr.蕭. ※Prof. Cheng has a natural compound for regulating the miRNA concentratin as fast as in 30 min.
SFL: Prof Cheng sugggested me to do the protien chip or pay more attention to the original material collection/ Prof Yan asked me if we have siRNA library for PTKs.
ENO1: 甲型烯醇酶 alphaEnolase. (Chimei 251 oral cancer patients. 106/251 with lymphonode metastasis)※ENO1/uPA/PA1 → MMP → galectin-3 cleavage → BrCA metastasis. ※ PAI promoter SNP polymorphism → higher incidence..? ※ Additional signals CXCL12, CCL2, STAT3※ENO1/Nrf2 and radiation resistance (IL6 by macrophage) → protect from chemotherapy and radiation therapy. Professor comments: inflammation is very close to hypoxia condition. VEGFR is closely related to metastasis. Question is: what is the resaronthat causese ENO1 to go up? is it totally the consequence of Hypoxia ? HIF1alpha?※the use of second ATG of ENO1 (48 kDa) produced MBP (37 kDa)
OVTA1/KIAAxxx/a Ser/Thr kinase: Ab from lung patient (turned out was a ovarian meta from lung) → serological screening of OVTA1 on ovarian cancer samples by using phage displaying system → N-terminus/Pro-rich/Ser/Thr Kinase domain/Ubi docking domain/regularatory domain (TPHGYAH motif identified by the Ab/phage displaying system) → expression level fo OVTA1 is associated with cell growth → si OVTA1, cell growth shut down/over-express OVTA1, cell growth increases → OVTA1 faciltates G1 progression※p21KO mouse 比同期的wild type 不易(or更容易)罹癌. OVTA1 may exert functions via Src. → OVTA1 gene-KO mouse with Dr.林SW at NTU. ※ENO1/inflammatory response and NF-KB
Nrf2/Keap1/ARF forms ubiquitination complex →IHC of Nrf2 expession level is high in oral cancer sample, but is heterogenesis (the use of wrong antibody?)※hypoxia and reoxygeneration※cisplatin/IR-resistant cells
B292 causes the increament of acetylated alpha tubulin, but inhibits tubulin polymerization ※a hybrid of J30 (taxol) and HDAC275※B292: for a series of cell line IC50 is about 50-100 nM but HUVEC is 42 uM, SAHA is 15.5 uM, 可是tube formation及 angiogenesis 又有被抑制. [me: alpha tubulin/HDAC6 expression in HUVEC, possibly be different from that of in other cell type??] low neuro toxicity in SH-SY5Y 250 nM no calcium influx.
PI3K/mTOR signaling pathways and H&N cancer: 90% EGFR is over-expressed in Euro/USA but no mutations was found; In Taiwan, EGFR amplicfication was found in 24~58% patients, EGFRVIII 42%. ※Dual mTOR and PI3K inhibitor:BGT226 Norvatis) (※No mutations was found in the PI3K or ?? regions of Taiwan HNSCC samples. Growth inhibition 12.5±5.1 nm. (Here comes a quiestion, Dr. Chang's 2 cell lines whose BGT226 sensitivity were not correlated with AKT phosphporylation) 覺得這部份的pathway 和 feed-back事件要搞清楚.
Secretome paper註載NPC and HNSCC and Chang-Gung Dr. Yu's Mac-2 in OEC-M1 and SCC-4.
3D culture and tumor micro-environment的模型建立. Valerie M Weaver的Webpage.
Dr.常DOH got funded, VEGF and SDF1/CXCR4 and signaling pathway biomarker with pancreatic cancer; desmoplastic (?) reactionin PDAC (=high fibrosis) → blood flow 差, 藥物無法到達tumor. ※ Tumor interstitial fluid pressure increases , vascular normalization by anti anti-angiogenesis. ※ Tumor blood flow interruption after radiation therapy strongly inhibts tumor regression. ※ radiation 造成細胞最嚴重傷害是在G2/M ※ shear stress/G2/M arrest/FAK activation/T84 cells/醫工組2008 PNAS paper/ ※ Prof Cheng's comment 搞清楚並模擬collagen I or IV/相對ITGs/ECM與coronial invasion system之關連.
Aldo-keto rectase (AKR) and redox GADD45-alpha ※ Nrf2 increases 則AKR increases, why? (because Nrf2 is related to ubiquitination system?) ※ c-Kit TK1 versus HSP90: AUY922 is a HSP90 inhibitor, AUY922 can efficiently suppress c-Kit and phosphorylated form of c-Kit, why?
Gemcitabine resistant cell lines ※ 2% O2 is a physiological hypoxia condition ※ HIF-alpha Yale 查一查..??
Treg and TAM in PDAC (a form of highly inflammatory disease)※ADM (acinar to ductal)→PanIN → PDA: K-Ras mutation (90%) → p16 loss (90%) → p53 inactivation (50-75%) → Smad4 loss (55%) ※Tumor associated macrophage (TAM) in BrCA and lung CA, the more infiltration of TAMs, the worse prognosis ※ conditional media from PBMCor THP-1 could stimulate the secretion of IL10, TGFb by TAM, to inhibits cancer cell proliferation. ※ Conditional media from cancer cells could stimulate TAM to secrete CCL22 ※ Lox-Stop-Lox (LSL)_Kras G12D x Pdx1-Cre
RT-PCR sequencing instead of genetic mutation check-u
TIEG1/KLF10: TGFbeta inducible early gene [Rev]. 與Sp1/Smad有關. Binding sites CCCTC??, could this be involved in DNA boundary process? HPRD shows the full-length one interacts with Sp1. ※Pim1 binds to a consensus motif pS/TP. ※Promoter of TIEG1 is highly methylated in oral cancer samples.
Kallikrein: KLK13/Cx 19/clustered gene family (proteases) [GeneID]: potential good biomarkers.
AXL inhibitor: 10-20 nM (IC50) Rigel Pharm... (CA, USA)
Combine Gencitabine and target thearpy (ex TKI, NFKB..) ※Aspirin is a IKKbeta inhibitor ※SAHA可contact夏醫師 ※first in man traial made in house: SCB01Q (BPRoL075)/CA4P tubulin polymerization and VDA (vascular disruption agent) ※CPT11在亞洲用藥要先genotype UGTA1*x6 nad UG1A1*28, Prof Yan suggested GenMED Chip {?} to analyze the genotype ※ Prof Cheng mentioned about Japan's CYP2D6.
Walter: MAGE A (CT1) gene family ※ 12 related gene members in the A family/located at Xq28, expressed during spermatogenesis. functions: binds to Skip, MAGE A11 can stablize ligand-free androgen, MAGE A and C-termunus can induces p53 dependent and independent apoptosis ※ MAGE A3: GSK 已將此分子列為tumor vaccine therapy, is a potential tumor marker present in 35-50% lung CA, 35% bladder CA, 74% in melanoma and HCC, 49% in Head and Neck cancers.
expression array of 40 N/T OSCC samples from Dr.蕭.
ShSSAT: growth inhibition escape. Illumina's miRNA and methylation array for 40 N/T OSCC samples from Dr.蕭. ※Prof. Cheng has a natural compound for regulating the miRNA concentratin as fast as in 30 min.
SFL: Prof Cheng sugggested me to do the protien chip or pay more attention to the original material collection/ Prof Yan asked me if we have siRNA library for PTKs.
ENO1: 甲型烯醇酶 alphaEnolase. (Chimei 251 oral cancer patients. 106/251 with lymphonode metastasis)※ENO1/uPA/PA1 → MMP → galectin-3 cleavage → BrCA metastasis. ※ PAI promoter SNP polymorphism → higher incidence..? ※ Additional signals CXCL12, CCL2, STAT3※ENO1/Nrf2 and radiation resistance (IL6 by macrophage) → protect from chemotherapy and radiation therapy. Professor comments: inflammation is very close to hypoxia condition. VEGFR is closely related to metastasis. Question is: what is the resaronthat causese ENO1 to go up? is it totally the consequence of Hypoxia ? HIF1alpha?※the use of second ATG of ENO1 (48 kDa) produced MBP (37 kDa)
OVTA1/KIAAxxx/a Ser/Thr kinase: Ab from lung patient (turned out was a ovarian meta from lung) → serological screening of OVTA1 on ovarian cancer samples by using phage displaying system → N-terminus/Pro-rich/Ser/Thr Kinase domain/Ubi docking domain/regularatory domain (TPHGYAH motif identified by the Ab/phage displaying system) → expression level fo OVTA1 is associated with cell growth → si OVTA1, cell growth shut down/over-express OVTA1, cell growth increases → OVTA1 faciltates G1 progression※p21KO mouse 比同期的wild type 不易(or更容易)罹癌. OVTA1 may exert functions via Src. → OVTA1 gene-KO mouse with Dr.林SW at NTU. ※ENO1/inflammatory response and NF-KB
Nrf2/Keap1/ARF forms ubiquitination complex →IHC of Nrf2 expession level is high in oral cancer sample, but is heterogenesis (the use of wrong antibody?)※hypoxia and reoxygeneration※cisplatin/IR-resistant cells
B292 causes the increament of acetylated alpha tubulin, but inhibits tubulin polymerization ※a hybrid of J30 (taxol) and HDAC275※B292: for a series of cell line IC50 is about 50-100 nM but HUVEC is 42 uM, SAHA is 15.5 uM, 可是tube formation及 angiogenesis 又有被抑制. [me: alpha tubulin/HDAC6 expression in HUVEC, possibly be different from that of in other cell type??] low neuro toxicity in SH-SY5Y 250 nM no calcium influx.
PI3K/mTOR signaling pathways and H&N cancer: 90% EGFR is over-expressed in Euro/USA but no mutations was found; In Taiwan, EGFR amplicfication was found in 24~58% patients, EGFRVIII 42%. ※Dual mTOR and PI3K inhibitor:BGT226 Norvatis) (※No mutations was found in the PI3K or ?? regions of Taiwan HNSCC samples. Growth inhibition 12.5±5.1 nm. (Here comes a quiestion, Dr. Chang's 2 cell lines whose BGT226 sensitivity were not correlated with AKT phosphporylation) 覺得這部份的pathway 和 feed-back事件要搞清楚.
Secretome paper註載NPC and HNSCC and Chang-Gung Dr. Yu's Mac-2 in OEC-M1 and SCC-4.
3D culture and tumor micro-environment的模型建立. Valerie M Weaver的Webpage.
Dr.常DOH got funded, VEGF and SDF1/CXCR4 and signaling pathway biomarker with pancreatic cancer; desmoplastic (?) reactionin PDAC (=high fibrosis) → blood flow 差, 藥物無法到達tumor. ※ Tumor interstitial fluid pressure increases , vascular normalization by anti anti-angiogenesis. ※ Tumor blood flow interruption after radiation therapy strongly inhibts tumor regression. ※ radiation 造成細胞最嚴重傷害是在G2/M ※ shear stress/G2/M arrest/FAK activation/T84 cells/醫工組2008 PNAS paper/ ※ Prof Cheng's comment 搞清楚並模擬collagen I or IV/相對ITGs/ECM與coronial invasion system之關連.
Aldo-keto rectase (AKR) and redox GADD45-alpha ※ Nrf2 increases 則AKR increases, why? (because Nrf2 is related to ubiquitination system?) ※ c-Kit TK1 versus HSP90: AUY922 is a HSP90 inhibitor, AUY922 can efficiently suppress c-Kit and phosphorylated form of c-Kit, why?
Gemcitabine resistant cell lines ※ 2% O2 is a physiological hypoxia condition ※ HIF-alpha Yale 查一查..??
Treg and TAM in PDAC (a form of highly inflammatory disease)※ADM (acinar to ductal)→PanIN → PDA: K-Ras mutation (90%) → p16 loss (90%) → p53 inactivation (50-75%) → Smad4 loss (55%) ※Tumor associated macrophage (TAM) in BrCA and lung CA, the more infiltration of TAMs, the worse prognosis ※ conditional media from PBMCor THP-1 could stimulate the secretion of IL10, TGFb by TAM, to inhibits cancer cell proliferation. ※ Conditional media from cancer cells could stimulate TAM to secrete CCL22 ※ Lox-Stop-Lox (LSL)_Kras G12D x Pdx1-Cre
RT-PCR sequencing instead of genetic mutation check-u
TIEG1/KLF10: TGFbeta inducible early gene [Rev]. 與Sp1/Smad有關. Binding sites CCCTC??, could this be involved in DNA boundary process? HPRD shows the full-length one interacts with Sp1. ※Pim1 binds to a consensus motif pS/TP. ※Promoter of TIEG1 is highly methylated in oral cancer samples.
Kallikrein: KLK13/Cx 19/clustered gene family (proteases) [GeneID]: potential good biomarkers.
AXL inhibitor: 10-20 nM (IC50) Rigel Pharm... (CA, USA)
Combine Gencitabine and target thearpy (ex TKI, NFKB..) ※Aspirin is a IKKbeta inhibitor ※SAHA可contact夏醫師 ※first in man traial made in house: SCB01Q (BPRoL075)/CA4P tubulin polymerization and VDA (vascular disruption agent) ※CPT11在亞洲用藥要先genotype UGTA1*x6 nad UG1A1*28, Prof Yan suggested GenMED Chip {?} to analyze the genotype ※ Prof Cheng mentioned about Japan's CYP2D6.
Walter: MAGE A (CT1) gene family ※ 12 related gene members in the A family/located at Xq28, expressed during spermatogenesis. functions: binds to Skip, MAGE A11 can stablize ligand-free androgen, MAGE A and C-termunus can induces p53 dependent and independent apoptosis ※ MAGE A3: GSK 已將此分子列為tumor vaccine therapy, is a potential tumor marker present in 35-50% lung CA, 35% bladder CA, 74% in melanoma and HCC, 49% in Head and Neck cancers.
Oct 26, 2009
Oct 24, 2009
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