Figure 3.

Transduction of different ALI culture types with F/HN pseudotyped LV

(A) Native EGFP detection from ALI cultures 14 days after apical delivery of 7.5 × 10⁷ transducing units (TUs) of LV.F/HN or LV.VSV-G expressing EGFP or mock-treated (buffer only). Representative stitched center-of-transwell images are shown (B-ALI, n = 4 donors; MucilAir and SmallAir, n = 2 donors; n = 2 biological replicates for each); scale bars represent 500 μm. (B) Quantification of EGFP (as percentage area above threshold fluorescence) from ALI cultures transduced or mock treated (buffer only) is shown. Each symbol represents a biological replicate, each shape represents a single donor, and the bar represents the median. ∗∗p = 0.004 (LV.F/HN-treated B-ALI versus MucilAir) and ∗p = 0.0286 (MucilAir vs SmallAir), as determined using Mann-Whitney U test. (C) Representative images of transwell cryosections show cell types transduced by LV.F/HN in B-ALI cultures, identified by co-localization (white arrows) of EGFP (green) with cell-type-specific antibodies (shown in red) to detect: basal (cytokeratin 5), ciliated (β-tubulin), goblet (mucin 5AC), and club (CC10) cells. Donor 1 is shown; nuclei are stained blue (DAPI); scale bars represent 50 μm. (D) Quantification of cell types expressing EGFP following transduction of B-ALI cultures with LV.F/HN is shown. Co-localization of EGFP and cell-specific markers was calculated from a minimum count of 100 EGFP-positive cells per B-ALI culture donor (n = 4) for each cell type. Each shape represents the cell donor; the bar represents the median. No club cells were identified in donor 2.