1. Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants 
Laura B. Gottschalk, Briana Vecchio-Pagan, Neeraj Sharma, Sangwoo T. Han, Arianna Franca, Elizabeth S. Wohler, Denise A.S. Batista, Loyal A. Goff, Garry R. Cutting 
Journal of Cystic Fibrosis 
Volume 15, Issue 3, Pages (May 2016)
DOI: /j.jcf
Copyright © 2015 European Cystic Fibrosis Society Terms and Conditions

2. Fig. 1
pFRT/lacZeo/ΔAGG is stably integrated onto chromosome 8. (A) Southern blot of hygromycin resistant clone using lacZeo specific probe. Arrow indicates band of genomic integration for clone. (B) Diagram of inverse PCR strategy to determine the genomic location of the integrated pFRT/lacZeo. Gray rectangle represents the pFRT/lacZeo plasmid integrated into the genomic DNA depicted by black lines. (C) Chromatogram of the inverse PCR product Sanger sequenced using the InvR primer. The resulting sequence contained a portion of pFRT/lacZeo along with 535bp of genomic DNA from chromosome 8. (D) Fluorescence in situ hybridization of CF8Flp cells in metaphase. Chromosome 8 was painted and appears green. A probe specific for pFRT/lacZeo is shown in orange. The inset shows a 3× magnification of the chromosome containing the positive signal for the pFRT/lacZeo probe.
Journal of Cystic Fibrosis  , DOI: ( /j.jcf )
Copyright © 2015 European Cystic Fibrosis Society Terms and Conditions

3. Fig. 2
The FRT site of CF8Flp cells is targetable and expresses functional CFTR. (A) PCR of genomic DNA from CF8Flp cells and CF8Flp cells containing GFP-CFTR to confirm integration. Primers to confirm integration were used for lanes one and two while lanes three through seven used primers spanning the length of CFTR cDNA. (B) FISH of CF8Flp cells containing GFP-CFTR using a probe specific to pFRT/lacZeo (C) Western blot of 40μg of whole cell lysate probed with antibody raised against GFP. CFBE cells transiently transfected with GFP-CFTR construct served as a positive control. (D) XY image of scanning disc laser confocal microscopy. CFTR is in red, GFP in green, and blue indicates DAPI stained nuclei. (E) XZ images of the cells show apical localization of the CFTR. Antibody specific to ZO-1 (red) hybridizes to the tight junctions of the polarized cells. (F) Representative tracing of electrophysiologic assessment of CF8Flp cells expressing WT-CFTR after sequential addition of 10μM forskolin and 10μM CFTR inhibitor 172 (G) Representative tracing from cells expressing G551D-CFTR after sequential addition of 10μM forskolin, 10μM Ivacaftor and 10μM CFTR inhibitor 172 (H) Average change in current after addition of forskolin or Ivacaftor plus forskolin (Mean±SEM, n=6 independent tracings).
Journal of Cystic Fibrosis  , DOI: ( /j.jcf )
Copyright © 2015 European Cystic Fibrosis Society Terms and Conditions

4. Fig. 3
The transcriptome of CF8Flp cells is stable between independently derived pools. (A) Diagram of timing for cell plating and harvesting for RNA-seq. Transepithelial electrical resistance (TEER) to monitor tight junction formation was measured every day until a resistance of ≥200Ω/cm2 was reached for three consecutive days, at which point cell lysate was collected and placed at −80°C. RNA was harvested from all frozen lysates simultaneously. B) Heat map of 13 housekeeping genes based on FPKM levels. Each row represents a gene while each column represents a cellular pool. C) Manhattan plots of CF8Flp parental cells compared to CF8Flp cells expressing either WT, F508del, or G551D CFTR. Red points indicate ≥3 SD from the mean of the statistic. CFTR and MUC4 are labeled for each plot.
Generated by CuffDuff software [17].
Journal of Cystic Fibrosis  , DOI: ( /j.jcf )
Copyright © 2015 European Cystic Fibrosis Society Terms and Conditions

5. Fig. 4
The CF8Flp transcriptome resembles that of primary bronchial epithelial cells. (A) Dendrogram derived from Jensen–Shannon distances calculated by using the top 50 genes with shared gene ontologies to CFTR. Data were obtained from the Sequence Read Archive and processed via the same RNA-seq pipeline as CF8Flp samples prior to normalization. (B) CFTR FPKM values from RNA-seq data (mean with 95% CI) (C) Manhattan plot of CF8Flp-WT-CFTR compared to FACS sorted lung epithelial cells. Red points indicate ≥3 SD from the mean of the statistic.
Journal of Cystic Fibrosis  , DOI: ( /j.jcf )
Copyright © 2015 European Cystic Fibrosis Society Terms and Conditions