Journal: medRxiv

Article Title: INTEGRATING GENOMIC AND FUNCTIONAL TESTING TO IMPROVE CFTR MODULATOR RESPONSE PREDICTION IN CHILDREN WITH CYSTIC FIBROSIS

doi: 10.1101/2025.07.09.25331152

Figure Lengend Snippet: A) Representative recordings and results from an individual participant. Changes in short-circuit current (ΔIsc) in differentiated human nasal epithelial cell (HNEC) culture from study participant number five. Cells were either untreated (0.01% DMSO vehicle) or pre-treated with a corrector(s) (Lumacaftor (LUM), tezacaftor (TEZ:T) or elexacaftor (E) plus TEZ for 48 hours). Functional CFTR expression was then measured by sequentially adding 100 μM apical amiloride (1. Amiloride), 0.01% apical DMSO vehicle control or 10 μM apical VX-770 (ivacaftor (IVA;I )), followed by 10 μM basal forskolin (3. Forskolin), 30 μM apical CFTR inhibitor (4. CFTRinh172), and 100 μM apical ATP (5. ATP). The basolateral-to-apical chloride gradient was used to measure functional CFTR activity. The i n vitro ΔIsc results for participant number five are displayed on the bar chart, with the dotted line indicating the wild-type reference level for our lab (as reported in Wong et al. 2022 42 ). CFTR activity is expressed as the inhibition of Isc by CFTRinh172 after activation by forskolin. Each dot represents an independent HNEC culture. Data are presented as mean ± SEM. B) ΔIsc in differentiated HNEC cultures to first modulator treatment. Bar chart showing ΔIsc (% of wild-type reference) in participants differentiated HNEC cultures in response to their first CFTR modulator treatment. CFTR activity is expressed as the inhibition of Isc by CFTRinh172 after activation by forskolin. Modulator response is calculated by subtracting baseline CFTR activity (DMSO) from modulator-treated results and reported as a percentage of normal (wild type). C) ΔIsc in differentiated HNEC cultures to subsequent modulator treatments. Bar chart showing ΔIsc (% of wild-type reference) in response to a subsequent CFTR modulator regimen. CFTR activity is expressed as the inhibition of Isc by CFTRinh172 after activation by forskolin. Changes are calculated by subtracting baseline CFTR activity (DMSO) from modulator-treated results and reported as a percentage of normal (wild type). Each bar represents the mean result of three replicate cultures per participant. Error bars represent the standard error of the mean (SEM). 113×233mm (300 × 300 DPI)

Article Snippet: Following a 30 minute stabilisation period, differentiated-HNEC cultures were treated with pharmacological compounds (in order): 100 μM amiloride (Sigma A7410, apical) to inhibit epithelial sodium channel (ENaC)-mediated sodium (Na + ) flux, vehicle control 0.01% DMSO or 10 μM IVA (VX-770; Selleckchem S1144; apical) to potentiate cAMP-activated currents, 10 μM forskolin (Sigma F6886, basal) to induce cAMP activation of CFTR, 30 μM CFTR inh -172 ( Selleckchem S7139, apical) to inhibit CFTR-specific currents and 100 μM ATP (Sigma A2383, apical) to activate calcium-activated chloride currents ( ).

Techniques: Functional Assay, Expressing, Control, Activity Assay, Inhibition, Activation Assay