Fig. 4: EGFR activation promotes MUC5AC expression by reducing the expression of claudin1.

a The 16HBE cells were stimulated respectively with HDM, LPS, TNF-α, EGF, and HBEGF for 24 h (representative blots from three experiments). b 16HBE cells were stimulated with various concentrations of HBEGF for 24 h, and the expressions of claudin1, E-cadherin, ZO-1, and occludin were detected by western blotting (representative blots from three experiments). c 16HBE cells were stimulated with HBEGF at indicated time points, and the expressions of claudin1, E-cadherin, and ZO-1 were detected by western blotting (representative blots from three experiments). d 16HBE cells were stimulated with HBEGF (20 ng/mL) for 24 h, and the expressions of claudin1, ZO-1, and E-cadherin were detected by immunofluorescence (Scale bar: 50 μm; original magnification:×400; representative images from three experiments). e 16HBE cells were stimulated with various concentrations of IL-13 for 24 h (representative blots from three experiments). f 16HBE cells were stimulated respectively with HBEGF (20 ng/mL), IL-13 (100 ng/mL), and HBEGF (20 ng/mL) + IL-13 (100 ng/mL) for 24 h (representative blots from three experiments). g, h 16HBE cells were incubated with AG1478 (10 μM) or with their vehicle, 0.1% dimethyl sulfoxideas a control, for 1 h. and were then treated with HBEGF or IL-13 for 24 h (representative blots from three experiments). i–k 16HBE cells were stimulated with HBEGF (20 ng/mL) for 24 h, and the expression of MUC5AC was detected, respectively, by immunofluorescence (Scale bar: 50 μm; original magnification: ×200; representative images from three experiments), western blotting (representative blots from three experiments), and qPCR (n = 6 per group). The relative intensity of MUC5AC was assessed by ChemiScope analysis software. l Differentiated cultures were treated basolaterally with HBEGF (10 ng/mL), transepithelial electrical resistance measurements were carried out on days 0, 7, 14, 21, and 28 following cell differentiation at the air-liquid interface, m–o The expressions of CLDN1, MUC5AC, CDH1, and OCLN in ALI cultures were determined by RT-PCR (n = 3 per condition). p 16HBE cells were transfected with pcDNA3.1‐CLDN1 or empty vector. After 6 h, the medium was replaced with RPMI 1640 containing 10% fetal bovine serum. After 24 h of continued culture, the cells were stimulated with or without HBEGF (20 ng/mL) for 24 h, and MUC5AC expression was detected by western blotting. Statistical comparisons was performed using two-way analysis of variance with Bonferroni’s multiple comparison and the unpaired two-tailed Student’s t-test with Welch’s correction (***P < 0.001; **P < 0.01; *P < 0.05; mean ± SD).