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Fig. 10. Disruption of adherent and tight junctions by HGF/SF was prevented by expression of membrane-anchored HB-EGF mutant. Experiments were performed as for the scattering experiment except that the cells were plated on glass coverslips. Forty-eight hours after the plating, cells were exposed to HGF/SF 20 ng/ml and cells were washed and fixed at 4, 8 and 12 hours after the HGF/SF treatment and subjected to immunofluorescent staining. (a) Adherens junction's integrity was assessed using anti-E-cadherin antibody followed by an anti-mouse FITC-conjugated antibody. 1-4: MDCK^con cells at 0, 4, 8 and 12 hours after HGF/SF treatment. The adherent junctions began dissociating 4 hours after HGF/SF treatment (see arrowheads) and had mostly disappeared after 8 hours, with the majority of E-cadherin translocated into the cytoplasm (see arrows), and after 12 hours post-treatment cells had begun to move apart. However, they were still connected with individual strands of adherent junctions (see double arrows). At this time-point most of the E-cadherin staining was lost possibly because of endocytosis. 5-8: MDCK^5aa cells at 0, 4, 8 and 12 hours after HGF/SF treatment. Although there was a small amount of cytoplasmic translocation of E-cadherin in response to HGF/SF treatment, the junctions were intact even after 12 hours after the treatment. Original magnificatiox400. (b) Tight-junction integrity was assessed using anti-ZO-1 antibody followed by anti-rabbit-rhodamine-X conjugated antibody. 1-4: MDCK^con cells at 0, 4, 8 and 12 hours after HGF/SF treatment The cytoplasmic translocation of ZO-1 was observed by 4 hours after HGF/SF treatment (see arrowhead) but the tight junctions were still largely intact. However, by 8 hours of HGF/SF treatment, tight junctions were markedly disrupted (see arrows) and by 12 hours post-treatment, almost all of junctional ZO-1 had translocated into the cytoplasm and cells had begun to move apart. However, they were still connected with strands of tight junctions (see double arrows). 5-8: MDCK^5aa cells at 0, 4, 8 and 12 hours after HGF/SF treatment. Although there was little cytoplasmic translocation of ZO-1 in response to HGF/SF treatment, the tight junctions were intact like adherent junctions in the MDCK^5aa cells even at 12 hours after the treatment. Original magnificatiox400.

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