Fig. 3

Alterations in NHBE ALI cultures induced by BMGLS. NHBE ALI cultures were supplied with BMGLS over the course of 3 weeks. (A) Increasing concentrations of BMGLS induced the formation of intra-epithelial structures (IES). IES, filled with acidic mucopolysaccharides, identified by using AlB staining (acidic mucopolysaccharides in blue; nuclei in purple; cytoplasm in pink). Black arrows indicate the opening of IES to the surface. Representative images are shown. Scale bars, 200 μm. Analysis was done by TEER (B) as well as histologically (C). Data between groups was compared using (B) One-way-ANOVA with Tukey’s or (C) Dunnet’s multiple comparison test. Significance was considered at p < 0.05 (*), p < 0.01 (**) and p < 0.0001 (****). (D) Representative IF image of proliferating cells (Ki67, green), apoptotic cells (cPARP, magenta) and nuclei (DAPI, white). No difference in the percentage of Ki67 positive or cPARP positive cells was detected after 3 weeks of BMGLS application. Differences were evaluated by One-way ANOVA with Tukey’s multiple comparison test. Statistical significance was considered at p < 0.05 (*). (E) Staining of NHBE ALI cultures following 0.6% BMGLS application with MUC1, MUC4, and PanCK illustrates epithelial origin and differentiation into a mucus producing REp. S100 stained negative disregarding degenerative processes in NHBE ALI upon BMGLS application. Scale bars, 200 μm