induces vimentin but reduces E-cadherin expression. Cultures were pretreated overnight with serum-free DMEM/12 at pH 7.4 and treated for 24 h with acidic serum-free medium at pH 6.8 and 7.4. The cells were subsequently fixed and incubated with antibodies to vimentin (A & B) and E-cadherin (C & D). FITC-labeled signals were detected by fluorescence microscopy. (A & C) Representative results were shown from three or more independent experiments. Immuno-positivity was caliculated and their positivity was shown as relative values compared to LLCm1 cells at pH
7.4. Bar, 50 μm. *P < 0.05; **P < 0.01; NS, not significant. E. Western blot analysis for E-cadherin. After incubation of cells at pH 6.8, as described above, cells were lysed and analyzed by Western blotting. CM was concentrated and analyzed to detect shedded E-cadherin level in the CM.