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Figure 6–

Figure 6–

Transforming growth factor (TGF)-β pre-treatment prolongs house dust mite (HDM)-induced disruption of epithelial junctions. a, b) 16HBE cells were cultured in LabTeks for 3 days, serum deprived for 4 h, pre-treated overnight with or without TGF-β (2 ng·mL−1) and subsequently stimulated with HDM (50 μg·mL−1) as indicated, in the presence and absence of AG1478 (1 μM). a) E-cadherin and b) zona occludens (ZO)-1 were detected by immunofluorescent staining. Representatives of three independent experiments are shown. c) 16HBE cells were grown on cover slips for 3 days, serum deprived for 4 h, pre-treated overnight with or without TGF-β (2 ng·mL−1) and subsequently stimulated with HDM (50 μg·mL−1) for 6 h. Electron microscopy analysis of tight junctions was performed as indicated (white arrows). Representative electron microscopy images are shown. d) Cells were grown on Electric Cell-Substrate Impedance Sensing (ECIS) arrays in duplicates for 3 days, serum deprived for 4 h, pre-treated overnight with or without TGF-β (2 ng·mL−1) and subsequently stimulated with HDM (50 μg·mL−1)/vehicle. Resistance was measured at 0 and 8 h upon HDM treatment at 400 Hz using ECIS. n = 6. *: p<0.05. Median values are indicated. Normal primary bronchial epithelial cells obtained from Lonza (Walkersville, MD, USA) e) and epithelial cells derived from bronchial brushings in asthma 16 were grown to confluence in LabTeks for 3–5 days, growth factor/hormone-deprived for 4 h, pre-treated overnight with or without TGF-β (2 ng·mL−1) and subsequently stimulated with HDM (50 μg·mL−1) or epidermal growth factor as indicated. E-cadherin and ZO-1 were detected by immunofluorescent staining. Representatives of three independent experiments are shown. f) Total cell lysates were prepared from bronchial asthma epithelial cells 16 and ZO-1 was detected by Western blotting (arrow). Representatives of three independent experiments are shown.