Corrector treatment increases CFTR activation response of nonsense variants in exon 22 that result in mature truncated CFTR. (A) Schematic of CFTR-Expression Minigene with abridged introns 21 and abridged intron 22 (EMG-i21-i22) constructed in pcDNA5FRT plasmid. CFTR exons are shown in boxes and two abridged introns in dashed lines. The location of each studied variant is shown relative to the CFTR exons and regions predicted to elicit NMD. (B) RT-qPCR showing relative steady state levels of CFTR transcript in HEK293 stable cells expressing wild-type EMG or EMGs with truncations at residue position, as indicated on the labels. Values were normalized to B2M . Mean ± SEM ( n = 3) measured in triplicates. P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001) when compared with CFTR mRNA abundance in cells expressing WT-EMG. (C) Steady state levels of CFTR protein from HEK293 cells transiently transfected with wild-type EMG or EMGs with different nonsense variants. 40 μg of total cell lysates were electrophoresed and IB was probed with anti-CFTR antibody-MM13-4 (EMD Millipore). (D) Representative IB showing sensitivity of CFTR to PNGaseF and Endo H. Mature complex glycosylated band is sensitive to PNGase only, whereas immature core glycosylated band is sensitive to both PNGase and EndoH. (E). Schematic illustration of the nonsense variants in the protein context showing their classification into two groups based on mRNA stability and protein maturity. Each nonsense variant truncates CFTR at intracellular loop 6 (ICL6) just before NBD2. (F) A representative Ussing chamber tracing of CFBE cells stably expressing S1196X-EMG. Short-circuit (I sc ) measurements were recorded in Ussing chambers after treatment of cells with 0.03% DMSO (vehicle) or 3 μM corrector compounds (lumacaftor/tezacaftor or both) for 48 h. ( G and H) Stacked bar graphs indicate effect of modulator treatment on CFBE (G) and MDCK (H) stable cells expressing different
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