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. 2019 Sep;61(3):322–331. doi: 10.1165/rcmb.2018-0326OC

Figure 4.

Figure 4.

Inhibition of BMP/TGF-β/SMAD signaling amplifies GCMH from early committed progenitor cells. (A) Human airway basal cells derived from a healthy donor were seeded on ALI. At Day 2, cells were cultured in control ALI medium alone, or ALI medium with IL-13 (20 ng/ml), or ALI medium with IL-13 (20 ng/ml) with A8301 (1 μM)/DMH-1 (1 μM) for another 14 days. ALI membranes were fixed to examine various airway epithelial cell markers. (B) Staining of AcTub (ciliated cells), CCSP (club cells), and MUC5AC and MUC5B (goblet cells) on ALI trans-sections. Scale bars: 10 μm. (C and D) Quantification of AcTub+ cells and CCSP+ cells (C) and MUC5AC+ cells and MUC5B+ cells (D) out of the total cells (total DAPI number) based on staining described in B. A total of three independent areas were imaged and quantified (mean ± SD; n = 3 independent imaging fields; **P ≤ 0.001). (E) Whole-mount staining of MUC5AC on ALI membranes. Scale bars: 100 μm. (F) Quantification of MUC5AC production in the experiment described in E. MUC5AC index was scored using ImageJ software based on the total area of MUC5AC immunopositivity out of the imaging area and calculated relative to the control without IL-13 stimulation. A total of three independent areas were imaged and quantified (mean ± SD; n = 3 independent imaging fields; **P ≤ 0.001). A/D = A8301 + DMH-1.