Fig. 2: The induction of Hic-5 depends on the activation of TGF-β receptor and ERK.

A TGFB1I1 mRNA expression progressively decreased as basal cells differentiated over the course of 21 days in ALI culture (mean ± SD, n = 2 transwells from a single HBE cell donor). B Representative western blots present that Hic-5 protein was prominently detectable in confluent basal cell layers on ALI day 0, and its expression decreased as the basal cells differentiated. Densitometry analysis was performed, and the fold change relative to GAPDH was calculated in arbitrary units (A.U.) (mean ± SD, n = 5 HBE cell donors). One-way ANOVA with Holm–Šídák post hoc correction (vs. ALI day 0): *p = 0.0426 (ALI day 7), *p = 0.0328 (ALI day 14), and *p = 0.0328 (ALI day 21). In A, B, each open circle represents an individual donor. C In well-differentiated HBE cells, Hic-5 protein increased in response to both mechanical compression and rhTGF-β1 treatment. Compression-induced Hic-5 protein expression remained constant for up to 72 h post-compression, whereas rhTGF-β1 stimulation progressively increased Hic-5 protein levels over 72 h. Densitometry analysis was performed, and the fold change relative to GAPDH was calculated in arbitrary units (A.U.) (mean ± SD, n = 2 HBE cell donors). D Representative western blots demonstrate that the compression-induced Hic-5 expression was attenuated by SB431542 (TGF-β receptor inhibitor) and U0126 (ERK inhibitor) but remained unaffected by AG1478 (EGFR inhibitor). E-cadherin and GAPDH were detected as loading controls. Densitometry analysis was performed, and the fold change relative to GAPDH was calculated in arbitrary units (A.U.) (mean ± SD, n = 2 HBE cell donors). E Representative western blots demonstrate that the TGF-β1-induced Hic-5 expression was attenuated by SB431542 and U0126. E-cadherin and GAPDH were detected as loading controls. Densitometry analysis was performed, and the fold change relative to GAPDH was calculated in arbitrary units (A.U.) (mean ± SD, n = 2 HBE cell donors). In C–E, each symbol represents an individual donor corresponding to the conditions indicated by the bars. F Representative western blots present the time course of phosphorylation of SMAD2, SMAD3, and ERK (p-SMAD2, p-SMAD3, and p-ERK) in HBE cells following stimulation with mechanical compression or TGF-β1. Compression prominently increased p-ERK levels as early as 15 min and sustained up to 60 min post-stimulation, but did not induce either p-SMAD2 or p-SMAD3. TGF-β1 substantially increased p-SMAD2 and p-SMAD3 within 30 min and sustained up to 120 min, while modestly increased p-ERK levels detected at 60 minutes (n = 2 HBE cell donors). Uncropped versions of the blots are provided as a Source Data file.