Peter van Mourik

24 Chapter 2 Figure 2. Protein and mRNA expression in R117H-CFTR organoids. (A) Representative images of the western blot analysis demonstrating CFTR C-band, CFTR B-band and HSP90 loading control protein expression. (B) CFTR protein (C-band) expression corrected for sample loading using HSP90 protein expression and normalized to protein expression in the wild type (WT) sample. (C) R117H-CFTR mRNA expression normalized for ACTB and YWHAZ gene expression and converted into relative quantities by normalization to organoid 1 (R117H-CFTR/R117H-CFTR). (D) Correlation analysis of normalized R117H-CFTR mRNA expression with normalized CFTR protein expression. Finally, we investigated the relations between FIS, mRNA and protein expression in R117H-CFTR organoids. Correlation was especially evident between 0.128 μM FIS and R117H-CFTR mRNA and protein expression (r=0.75, p<0.01 and r=0.74, p<0.01, respectively, Figures 3A and 3B). This correlation persisted with the 0.05 =.01, respectively, Figures 3A and 3B). This correlation persisted with the 0.05 μm FIS results and mRNA and protein expression, albeit less strongly (r=0.57, p=0.04, and r=0.56, p=0.04, Supplementary figure 1B and 1C, respectively). VX-770-treated FIS at 0.05 μM forskolin demonstrated a correlation with mRNA (r=0.72, p<0.01, Figure 3C) and protein (r=0.72, p<0.01, Supplementary figure 1D) expression, which was also present after correcting for vehicle-treated organoid swelling (r=0.69, p<0.01, Figure 3D and r=0.69, p<0.01, Supplementary figure 1G, respectively). FIS of VX-770-treated organoids with 0.128 μM forskolin did correlate with mRNA and protein expression (r=0.57, p=0.03, and r=0.6, p=0.02, Supplementary figure 1E and 1F, respectively) but after correcting for vehicle-treated organoid swelling these correlations were lost (see Supplementary figure 1H and 1I).