Progress Report: 1. The stability of EBV Rta a. the expression level of RTS-15 and Flag-Rta is similar but slightly higher than Rta-V5. b. Rta expression level of 6-hr transfection is blocked by MG115 and fragmented into smaller size, and the fragments are stablized by MG115 treatment.2. The expression level of Akata-EGFP/EBV and Akata-p2089 is […]
http://www.nature.com/nrc/journal/v11/n2/full/nrc3012.html It is generally assumed that tumours evolve through a progressive acquisition of mutations in the genome that allow cells to evade apoptosis, proliferate, invade and metastasize. However, in some instances one single event can lead to multiple coexisting mutations — the loss of telomeres results in end-to-end chromosome fusions that lead to chromosomal rearrangements.
報告進度:1. 利用外送PCDNA3-Flag CDK9, PCMV-Flag2-DN-CDK9及PIRES-hrGFP-cyclin T1進行K-RTA的in vitro kinase assay, 初步看到KRTA被CDK9磷酸化,而DN-CDK9則不具有這樣的能力2. silence DDR1後對於oral cancer cells生長曲線的影響希望快快將data補起來!! 加油!!
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報告英國Sahai先生兩篇collective cell migration的paper。2007年這篇是他們建立3D culture system研究squamous cell carcinoma (SCC)的collective invasion，他們發現，cancer associated fibroblast具有帶領一整群SCC invasion的能力。而cancer associated fibroblast會造成matrix remodeling及”track”的形成，以幫助SCC invasion，而這是經由integrin a3、a5、Rho-ROCK路徑。 2011這篇延續2007年的發現，進一步詳盡探討SCC的collective invasion，他們發現要發生collective invasion時，cancer cell的actomyosin (包含F-actin, phospho-MLC及MyosinⅡa)會reorganization。他們觀察到癌細胞與細胞間的actomyosin的活性都是明顯下降的，並進一步發現DDR1會參與actomyosin活性的調控，而DDR1調控collective invasion與它已知的ligand ”collagen”及它的kinase activity沒有太大的關聯性。利用GST-pull down發現DDR1會與Par3及Par6的PDZ domain形成complex，並進一步影響RhoE的分佈，而影響ROCK driven的actomyosin活性，因此，一但阻斷DDR1/Par3/Par6，SCC的collective invasion則會被中斷。本篇提供了DDR1/Par3/Par6在SCC的collective invasion時可能扮演的角色。
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報告進度: 1. 偵測Dox-treated TW01-EREV-26/27的 a. viral particles, 似乎隨時間增加, 但infectivity待測 b. viral protein expression pattern比EREV8稍快 c. 27的viral particles production比26高 d. 此次的NaB效果遠比Dox低, 可多試NA或EREV8當positive control, 確定NaB效果 e. 細胞數少的induction efficiency較好 2. ChIP的sonication條件: fixation mode: suspension cell conc: 1.5 x 10^7 cells/ml, sonication vol: 150 ul/reaction sonication cycle: 30″ ON, 30″ OFF, for 10 cycles 3. RTS-15與Flag-Rta送入293細胞後的表達量相當, 但加MG115之後未stablize Rta的量
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報告進度(1) TW03Tet有10株細胞株，TW05Tet有8株細胞株，已凍管且收好cell lysates等待Western analysis確認。 (2) 目前要進行的實驗如下a. 將不同稀釋倍數(0、1、1/2、1/4、1/8、1/16)的pAS-EGFP lentivirus送入293中，48小時後，計數細胞存活及死亡比率，同時跑FLOW。(這次infection時，不從細胞上移除含有polybrene的medium)b. 以shLuc、shOGT、shMGEA5的lentivirus送入ERKV中，24、48、72小時後，收lysates進行WB確認目標蛋白質是否有減少。c. 將OGT Ab的條件抓好。
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報告Hart新作：(1) 以chemoenzymatic assay及抗體確認histone有O-GlcNAcylation(2) 以LC MS/MS定義出histone上有被O-GlcNAc修飾的位點：Ser47 of H4、Ser36 of H2B和Thr101 of H2A(3) Heat shock(45℃，1hr)及後續的recovery的時候，發現histone的O-GlcNAc程度逐漸提升，同時OGT活性也有緩慢上升(4) 以MNase chromatin-sensitivity assays檢測發現Heat shock及後續的recovery時候，Chromatin(染色質)會condense(5) 過量表現OGT會比過量表現GFP的細胞的染色質更加condense
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1. 成功移除OEC-M1內的mycoplasma2. 比較多株口腔癌細胞DDR1 autophosphorylation的情形 初步結果顯示台灣地區口腔癌細胞株的DDR1 tyrosine phosphorylation的程度較高3. 一系列K-RTA vs. CDK1,2,6,9的in vitro kinase assay 仍須再試條件 讓結果更明顯 希望會成功!!!
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原文Nature.Next, we performed immunohistochemistry (IHC) on approximately 115 human tissues ranging from benign nevi to metastatic melanoma (tissue set 1). mH2A2 antibody was used for IHC, as it produced clear nuclear staining, and tissues were independently scored by two blinded dermatopathologists with excellent interobserver consistency. IHC demonstrated that although mH2A2 is abundantin melanocytes of benign