Résumé
Objective To investigate the effect of exogenous NPM mA to the subcellular localization of AKT and its significance in the proliferation in leukemia cells.Methods The co-immunoprecipitation assay was used to detect the interaction between NPM mA and AKT.The subcellular localization of NPM mA and AKT was observe by using the immunofluorescence experiment.The cells proliferation potential was assessed by CCK-8 assay.Results NPM mA could be combined with AKT in K562 cells.The immunofluorescence showed that NPM mA was mainly distributed in the cytoplasm.After co-tranfecting NPM mA and AKT into HEK293T cells,AKT was transformed from the dispersive distribution to cytoplasma.Additionally,the in vitro proliferation ability at 72 h in the K562 group was significantly enhanced compared with the K562 c1 and K562 mA groups(P＜0.01).After treating with AKT inhibitor(AKT inhibitor Ⅳ,AKT Ⅳ) for 48 h,the proliferation in the K562 c1 and K562 wt groups was remarkably inhibited,but which in the K562 mA group could still maintain growth(P＜0.01).Conclusion Exogenous NPM mA can change the subcellular localization of AKT,moreover regulates the in vitro malignant proliferation of leukemic cells.
Résumé
Objective To observe the expression and localization of D J-1 in renal fibrosis, and to investigate the expressions of E-cadherin, vimentin and the level of β-catenin tyrosine phosphorylation in human tubular epithelial cells. Methods In vitro, the human tubular epithehal cells (HKC cell line) were cultured with 10 μg/L TGF-β1 for 72 h. The protein expressions of E-cadherin, vimentin and DJ-1 were measured by Western blot. RT-PCR was used to detect the expression of D J-1 mRNA. The intracellular distribution of DJ-1 was observed by confocal microscope. In vivo, Masson stain was used to evaluate the level of renal fibrosis. The expression and disposition of DJ-1 in renal tissue were detected by immunohistochemistry. HKC cells were transfected with pEGFP-N1-DJ-1 via lipofectamine 2000. The efficiency of transfection was detected by fluorescence microscope. The expressions of DJ-1, E-cadherin, vimentin and β-catenin tyrosine phosphorylation level in the transfected cells were detected by Western blot. Results The expressions of DJ-1 protein and DJ-1 mRNA were up-regulated in renal tubular EMT cells. Most of DJ-1 protein localized in cytoplasm, and some was in nucleus. After stimulation by TGF-β1, the expressions of DJ-1 protein both in cytoplasm and nucleus was greatly increased, especially in nucleus. In vivo, renal tissue expressed DJ-1 in tubular epithelia, but little expression in glomeruli. In renal tissue from 5/6-nephrectomized rots, DJ-1 expression was greatly increased. In the DJ-1 transfectants, the expressions of DJ-1, vimentin and β-catenin tyrosine phosphorylation level were up-regulated, but E-cadherin expression was suppressed. Conclusion The increased expression of DJ-1 may promote renal fibrosis.