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Objective To investigate the mechanism of xCT on tumor metastasis in breast cancer cell MDA-MB-231. Methods Wound scratch assay and Transwell assay were performed to evaluate the effect of disruption and knockdown of xCT on cell migration and cell invasion in breast cancer cell MDA-MB-231 .Western Blot and RT-PCR were used to detect the expression levels of autophagy and EMT related markers in breast cancer cell MDA-MB-231 after treatment with sulfasalazine (SASP), an inhibitor of xCT activity and SLC7A11-RNAi.Results Both the scratch assay and the transwell migration assay showed that inhibition of xCT reduced the motility of MDA-MB-231 .The expression level of autophagy related protein LC3-Ⅱ/LC3-Ⅰwas elevated, the protein level of transcription factor Snail was down-regulated, while the mRNA level of Snail did not change in xCT inhibited MDA-MB-231 cells compared with MDA-MB-231 cells.Epithelial marker E-cadherin was up-regulated but mesenchymal marker Vimentin was down-regulated when xCT was deficient.Con-clusion Our current studies show that xCT is an endogenous regulator of tumor growth and metastasis in MDA -MB-231 and the expression level of xCT determines the phenotypes of MDA-MB-231 cells in invasion and migration in vitro.Inhibition of xCT can activate autophagy , induce the degradation of Snail ,and attenuate the EMT process in highly metastatic MDA-MB-231 cells.
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Objective To identify the mechanisms underlying xCT deficiency by high-resolution proteomic analysis of differential protein expression in Sut and wild melanocytes .Methods The proteins,extracted from Sut and wild melano-cytes,were analyzed by two dimensional electrophoresis and PDQuest software .Subsequent MALDI-TOF mass spectrometry analysis was carried out .The protein identification was based on peptide mass fingerprint combined with the pI and the rela -tive molecular mass ( Mr) .Sequence coverage was performed with the Peptldent software on the NCBnlm website .Also,the autophagy marker protein LC3-Ⅱ, and the autophagic cell death marker protein Beclin 1 were detected by Western blotting . Results and Conclusion Twenty apparently upregulated or downregulated proteins were identified .Strikingly, important modifications in regulators of trafficking and organization of vesicles and autophagy ( Anxa3; Hist 1h2bk, NDRG1, and CaM) and in regulators of invasion and metastasis of carcinoma (S100A-4;S100A-6 and vimentin)are likely to account for dysfunctions in cell viability and cell-extracellular adhesion .These results indicate that the proteins regulating autophagy , vesicles trafficking and MAP kinase related pathways are activated and play a role in xCT-deficiency .
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ObjectiveTo analyze the autophagy of mouse melanoma B16 cells induced by TNF-ct.MethodsMouse melanoma B 16 cells treated with TNF-α were used in this study.The expression levels of LC3- Ⅱand Beclinl were measured by western blot and mRNA levels of autophagy related gene atg5,atg7 and atgl2 were measured by reverse transcription polymerase chain reaction (RT-PCR) after TNF-α treatment.ResultsThe viability of B16 cells were obviously inhibited after incubation with TNF-α.The expression levels of autophagy related protein LC3- Ⅱ and Beclinl were elevated in TNF-α treated B16 cells compared with in control B16 cells.The mRNA expression levels of autophagy related gene Atg5,atg7 and atg12 showed consistent up regulation in TNF-a treated B16 cells compared with in control B16 cells.ConclusionTNF-α can induce autophagic cell death in B16 cells.
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Objective Predicting protein structural class is the basis for predicting protein spatial structure,so it is important to improve the prediction accuracy of protein structural class.Methods We proposed 3-state and 8-state Hidden Markov model (HMM),and applied these HMMs to the prediction of protein structural class,respectively.We evaluated their accuracy on two different datasets through the rigorous jackknife cross-validation test.Results Prediction ability of 8-state HMM and 3-state HMM to all α class were excellent,the prediction accuracy of 3-state HMM even reached above 95%.Compared with Chou data set,the prediction accuracy of Zhou data set for all β class and α/β class of was improved,while overall prediction accuracy increased by 2%.Conclusion HMM is an effective method to predict protein structural class.
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Objective To investigate the mechanism of Sut melanocytes growth inhibition in response to xCT -deficiency. Methods TTotal proteins were extracted from xCT-deficient Sut melanocytes and wild melanocytes, respectively, and were separated by 2-dimensional electrophoresis. Altered expression profile of proteins of Sut melanocytes was analyzed by PDQuest software and compared with that of wild melanocytes.Proteins with significant change were chosen to be identified by mass spectrometry and database query.Results Twenty proteins in Sut melanocytes altered significantly compared with wild melanocytes. Ten of the proteins were up-regulated, while the other tens were down-regulated. Four proteins from both up-regulated and down-regulated were identified respectively: up-regulated proteins were Tubulin alpha-1b, S100-A6,Nucleoside, S-formylglutathione, and down-regulated proteins were Calumenin,NDRG1 ,DPYSL2, 14kDa unknown protein. Conclusion The identification of the xCT-deficiency related proteins may provide supporting evidence for the mechanism research of Sut melanocytes' growth inhibition caused by xCT-deficiency.
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Objective Alzheimer's disease(AD) is a common and devastating disease and there is no readily available biomarker to aid diagnosis or to monitor disease progression. This study is to further understand the pathogenesis of Alzheimer's disease and seek new biomarkers for AD. Methods AD rats made by Aβ1-40 were assessed using the Morris water maze method. Sera of two groups were collected and albumin, IgG and sodium chloride were removed from the serum before concentration. Two-dimensional electrophoresis(2-DE) was performed on serum protein. Altered proteins were identified by matrix assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS). Results Compared with untreated samples, 10 altered proteins were found through 2-DE and all of them were down-regulated of which 2 were identified by MALDI-TOF-MS to be plasma retinol-binding protein precursor and complement component 4, gene 2 (C4b). Conclusion C4b may be the biomarker of AD and it may be helpful to understand the pathogenesis of AD.