Figure 3.

Characterization of primary differentiated NHBE:CFBE cell mixed culture and CFBE cell models

(A) Schematic of the NHBE:CFBE cell mixed culture model. CFBE and NHBE cells from male (XY) or female (XX) donors were combined in ratios of 10%, 25%, 50%, and 75% NHBE:CFBE cells and seeded onto semipermeable membranes. (B) Confocal microscope images (maximal projection) of a representative 50% NHBE:CFBE cell, stained with phalloidin (red, F-actin), anti-β-tubulin (yellow, cilia), DAPI (blue, nuclei), and composite image. Scale bars are depicted in the bottom right corner. (C) Relative proportions of AMEL-X and AMEL-Y regions in genomic DNA from three different mixes of male (M) and female (F) NHBE or CF donors post functional analysis, as determined via ddPCR. Data were normalized to values from 100% male or 100% female cultures, and samples were run in duplicate (n = 28). Each data point represents the mean ± SD (some error bars are smaller than the symbol height). (D) Schematic of the lentiviral constructs used for CFTR transduction. (E) Confocal microscope images of GFP CFTR-transduced CFBE cell cultures immunostained with phalloidin (F-actin, red), anti-β-tubulin (cilia, yellow), GFP (transduced cells, green), and composite image with DAPI (blue). (F) Relationship between percent seeded transduced cells and percent final transduced (4 h prior to functional testing) for the different CFTR cDNAs. The ratio of percent final transduced to percent seeded transduced (%T/%S) for each CFTR is given in a table below the graph.