Fig. 2

NE degrades cell-associated E-Cad and generates a distinct extracellular fragment. Confluent 16HBE cells were left untreated or treated with varying concentrations of purified NE (0, 2, 20, or 200 nM) for 6 h. Next, equal protein aliquots from culture supernatants and cell lysates (10 μg) were subjected to SDS-PAGE and immunoblotting using antibodies raised against N- and C-terminal parts of E-cad respectively. a, left panel. Anti-E-cad N-terminal antibody revealed a progressive increase of a distinct N-terminal fragment of about 80 kDa that paralleled the increase of NE concentration. a, lower panel, densitometric analysis confirms increased levels of E-cad fragment. Data are mean values ± SD. *p < 0.05; Kruskall-Wallis test. b, right panel. Loss of intact E-cad was achieved with NE at 200 nM. Of note, anti-E-cad C-terminal antibody detected varying fragments. NE inhibition with SLPI (400 nM) prevented degradation of cell-associated E-cad. b, lower panel, densitometric analysis found low levels of intact E-cad when cells were exposed to NE alone. However, preincubation of NE with SLPI prevented considerably such degradation. Data are mean values ± SD. *p < 0.05; Mann–Whitney test. c, left panel. Immunoblotting analysis of condition media from B with an anti-E-cad N-terminal part confirmed the generation of the distinct 80 kDa degradation product, which paralleled the loss of intact E-cad. c, lower panel, densitometric analysis found increased level of E-cad fragment concomitant with low levels of intact E-cad when cells were exposed to NE alone in B. However, preincubation of NE with SLPI prevented considerably E-cad degradation. MW standards are on wright. a.u., arbitrary unit. Data are mean values ± SD. * corresponds to p < 0.05 for cleaved versus intact E-cad; Mann–Whitney test. Experiments were repeated three times with similar findings