Fig. 5.

Differences in the functional and morphological properties of ALI multilayered mono- and co-cultures. a and b Percentage (%) of a live A549 cells and b total ATP levels detected in ALI multilayered mono- or co-cultures. Significant differences were detected among the ATP levels of the two cultures (one-way ANOVA with Dunnett post-test). c Western blotting analysis of the expression of surfactant-associated protein-D (SP-D) in ALI multilayered mono- or co-cultures. Both SP-D monomeric subunit were detected: the dominant form at 40 kDa and the additional subunit at 50 kDa (glycosylated SP-D). Abbreviations “n₁” and “n₂” indicate different biological replicates. GADPH expression is also reported as protein loading control. d Representative LSCM images of (from top to bottom) ALI multilayered mono- and co-cultures. ALI MCCs were stained for (from left to right) surfactant-associated protein-A (SP-A) and -C (SP-C) (both in green). The uneven staining was probably associated to the poor ability of A549 cells to produce surfactant-associated proteins. Cells were also stained for F-actin (in red) and nuclei (in blue). Z-stack images of the cultures were reconstructed and are shown in orthogonal view (ortho-view) or as three-dimensional projections. Scale bars: 10 μm (objective lens: 63×)