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. 2022 Sep 15;132(18):e154571. doi: 10.1172/JCI154571

Figure 4. CC-90009 reduces ENaC expression and rescues CFTR by promoting readthrough and full-length CFTR production.

Figure 4

(A) Relative SCNN1A, SCNN1B, SCNN1G, and CFTR mRNA levels by RT-qPCR in a panel of W1282X/W1282X cell lines and parent cells. Data were analyzed using a linear mixed-effects model with the donor as a random effect factor and are presented as the mean ± SD. n = 2–6 for each cell line. Post hoc comparisons were performed using the general linear hypothesis test. *P < 0.05 and ***P < 0.001. (B) Western blot for ENaCa in UNCCF9T parent cells treated with escalating doses of CC-90009. ENaCa expression normalized to GAPDH and relative to the 0.1% DMSO control is quantified below. Cells were grown in Vertex ALI media to increase the level of ENaC expression for detectability by Western blotting. (C) Western blot for eRF3a in a panel of W1282X/W1282X cell lines and parent cells treated with 0.1% DMSO or 0.1 μM CC-90009. eRF3a expression normalized to GAPDH and relative to the paired DMSO control is quantified below. (D) CFTR IP–Western blot (top) of the UNCX2T cell line (W1282X/W1282X) treated with 0.1% DMSO or 0.1 μM CC-90009 or the UNCNN2T non-CF cell line. A nonspecific band was observed in all samples and is indicated by a pound sign. CFTR expression normalized to actin and relative to the UNCNN2T non-CF control is quantified below. The same blot was reprobed for eRF3a (bottom). eRF3a expression normalized to actin and relative to the DMSO control is quantified below. All samples were run on the same blot and imaged at the same intensity level.