RESUMEN
AIM:To identify and analyze tyrosine-phosphorylated proteins regulated by protein tyrosine phos-phatase-like A domain containing protein 1 ( PTPLAD1) in colon cancer cells by phosphoproteomics.METHODS: The expression of PTPLAD1 in colon cancer cell line HCT-116 was knocked down by small interfering RNAs, and the differenti-al expression of tyrosine-phosphorylated proteins in response to the konckdown of PTPLAD1 in HCT-116 cells was identified by stable isotope labeling with amino acid in cell culture ( SILAC) , coupled with the tyrosine phosphorylation antibody im-munoprecipitation and LC-MS/MS analysis.The Ingenuity Pathway Analysis ( IPA) software was employed for bioinformat-ics analysis on the differentially-expressed proteins.RESULTS:A total of 20 differentially-expressed tyrosine-phosphoryla-ted proteins were identified by MS, including 8 markedly up-regulated and 10 evidently down-regulated proteins.IPA soft-ware suggested that these proteins were mainly associated with the disease of cancer, tissue development and function, and cell death and survival.CONCLUSION:We successfully identified PTPLAD1-regulated differentially-expressed tyrosine-phosphorylated proteins in colon cancer cell line HCT-116.Our analysis suggests that PTPLAD1-regulated proteins in colon cancer are closely correlated with colon cancer.
RESUMEN
[ ABSTRACT] AIM:To investigate the molecular mechanism and the immunosuppressive phenotype of macropha-ges under long-term exposure to lipopolysaccharide ( LPS) .METHODS:We used Ficoll-Hypaque density gradient centri-fugation combined with MicroBeads Separation Kits to separate peripheral blood mononuclear cells from human blood, and then induced the monocytes into macrophages.We observed the morphology of the macrophages by treating the cells with LPS for 48 h, in comparison with a negative control and IFN-γtreatment.ELISA was used to detect the levels of cytokines, such as IL-10, IL-12, IL-6 and TNF-α, and flow cytometry was used to detect the expression of the surface molecules (HLA-DR, CD14, CCR7, HLA-ABC and CD40).To observe the effect of macrophage on T cell proliferation, co-culture experiment was carried out for 6 d.Real-time PCR was used to validate the expression levels of molecules related to MyD88-independent pathway in Toll-like receptor 4 ( TLR4) signal pathway.RESULTS:The antigen-presenting ability of the macrophages was reduced and the IL-10 expression level was increased after the cells were treated with LPS for 48 h. We observed a poor proliferative capacity of CD8 +T cells after co-culturing of LPS-induced macrophages with CD3+T cells for 6 d.The results of real-time PCR indicated that TRIF, IRF3 and CIITA were down-regulated in LPS-induced macropha-ges.CONCLUSION:We successfully established a macrophage model in vitro and observed that LPS-induced macropha-ges into an immunosuppressive phenotype with poor CD8 +T cell proliferative capacity, in which MyD88-independent TLR4 signaling pathway was impaired.