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. 2023 Jan 14;31(4):970–985. doi: 10.1016/j.ymthe.2023.01.014

Figure 5.

Figure 5

Rescue of CFTR function in CF patient cells and in 16HBE14o− cells carrying a nonsense mutation in both Cftr alleles

(A) CFTR function was assessed in CF patient cells by means of an SPQ assay after DMSO (red), G418 (green), or DAP (blue) treatment. Arrows indicate addition of the forskolin-cAMP cocktail to the cell medium. The tested genotypes are W1282X/W1282X (patient 1), G542X/G542X (patients 2 and 3), and WT (lower right). (B) Sample single-channel activity of the CFTR Cl− currents in the 16HBE14o− expressing wild-type (WT) CFTR without (left) or following pretreatment with 40 μM forskolin and 400 μM IBMX for 30 min, prior to patching the cells (right). Note the absence of activity in patches made to non-stimulated cells and the presence of a characteristic CFTR activity following forskolin/IBMX treatment. (C) Single-channel activity of CFTR Cl− currents in 16HBE14o− cells expressing mutant CFTR. To stimulate CFTR currents, cells were pretreated with 40 μM forskolin and 400 μM IBMX for 30 min, prior to patching the cells. Sample traces were acquired in excised outside patches of forskolin/IBMX-pretreated cells expressing, from the top to the bottom, W1282X, G542X, and R553X CFTR. Note the absence of activity in the non-treated (left side) cells and presence of the characteristic CFTR activity in the cells treated with DAP. C and O denote closed and open states of the ion channel. (D–F) Whole-cell currents in isolated cells from WT (D), HO DAP (E), and HO DMSO (F) mice. Whole-cell currents were acquired in NMDG-Cl-based solutions as described. After whole-cell mode was established, the cells were left undisturbed for 5 min to allow replacement of intracellular K+ and Na+ with NMDG, to register basal current levels with Cl− as the major ion current carrier. This was followed by application of 40 μM forskolin and 400 μM IBMX to stimulate CFTR activity. Panels compare representative IV (current-voltage) relationships (left) as well as the time course of the CFTR activity stimulation with forskolin/IBMX (right). Note an increase in current following forskolin/IBMX application in WT and HO DAP mice and the absence of the effect in the HO DMSO case.