METHODS
Cell culture. The CF bronchial epithelial (CFBE) cell line stably expressing F508del-CFTR was cultured as before (Canato et al.,2018). CFBE cells stably expressing mCherry-Flag-CFTR (WT, F508del, DD/AA variants or carrying G550E, R1070W, 4RK in cis with F508del) were cultured and CFTR expression was induced with doxycycline (Dox) 1 µg/mL as described (Botelho et al., 2015). Fischer rat thyroid (FRT) epithelial cells stably expressing CFTR variants (wt, G85E, R334W, T338I, R347P, F508del, V520F, S549F, G551D, M1101K, N1303K) were cultured as before (Turner et al., 2020). All cell lines were maintained in a humidified incubator at 5% CO2 and 37°C, except during low temperature experiments, in which cells were incubated at 27°C for 24h.
Chemicals . All reagents were of the highest purity available. Corrector compounds were either commercially obtained: RDR01752 (STK001879, Vitas-M Lab., IL, USA), VX-809, VX-661 and VX-770 (S1565, S7058 and S1144, Selleckchem, TX, USA) or from CFFT: C18. Correctors were diluted in dimethyl sulfoxide (DMSO) and added to cells diluted in 1% FBS supplemented medium at indicated concentrations: 1 to 20µM RDR01752, 1 or 3.7µM VX-809, 5µM VX-661, 5µM C18, 3µM VX-770. Other reagents (all from Sigma, MO, USA, in DMSO solutions) were (final concentrations, unless otherwise stated): 2µM forskolin (Fsk), 50µM genistein (Gen), 100µM 3-Isobutyl-1-methylxanthine‎ (IBMX) and 30µM CFTR channel inhibitor CFTRInh-172, (Inh172).
Western blotting (WB). Whole-cell lysates were subjected to SDS-PAGE 7% (w/v) gel analysis followed by CFTR detection using monoclonal anti-human CFTR antibody (596 [1:3000] from CFFT) as previously (Farinha et al., 2013). Anti-α-tubulin antibody (1:10,000, Sigma) was used as a loading control. CFTR quantification was as described (Canato et al., 2018) and its processing obtained by the ratio between the mature form and total CFTR (mature and immature forms).
Immunostaining. Twenty-four hours after seeding stably expressing mCherry-Flag-CFTR CFBE cells (carrying wt-, F508del-, DD/AA- or -G550E-F508del, R1070W-F508del, 4RK-F508del-CFTR) onto 384-well plates, compounds were concomitantly administered with Dox. After 48h, cells were immunostained and CFTR PM expression quantified as described (Botelho et al., 2015).
HS-YFP assay on the plate reader . Measurements of CFTR activity were carried out on CFBE cells expressing both F508del-CFTR and HS-YFP (YFP-H148Q/I152L) as described (Sondo et al., 2011). The assay consists of a continuous 14-s fluorescence read-out 2s before and 12s after injection of an iodide-containing solution (PBS with I- at final concentration 100mM). Fluorescence quenching rate of I- influx was determined for the final 11s of the data for each well and was fitted to an exponential function to extrapolate initial slope (Sondo et al., 2011).
FLIPR membrane potential (FMP) assay. Measurements of FMP to assess CFTR function were performed in FRT cells expressing various CFTR mutants as described (Carlile et al., 2018 ).
Micro-Ussing chamber measurements. Transepithelial electrical resistance (TEER) of CFBE cells growing on Snapwell inserts was measured as before (Awatade et al., 2015). Briefly, monolayers with resistance ≥450Ωcm2 were mounted in micro-Ussing chambers with perfusion for recordings under open-circuit conditions as described (Awatade et al.,2015). Changes in transepithelial voltage (Vte) were continuously recorded and equivalent Fsk/IBMX-stimulated short-circuit currents (Ieq–sc) were calculated by Ohm’s law from Vte and Rte(Ieq–sc=Vte/Rte). For FRT cell lines, transepithelial voltage was measured at 37°C with continuous stirring by gassing with 95% O2 and 5% CO2 as described (Turner et al., 2020).
Organoid swelling assay. The forskolin-induced swelling (FIS) assay was performed as described (Dekkers et al., 2016). Twenty-four hours after seeding, organoids were stimulated with Fsk with or without a potentiator and live-cell imaging was performed using bright field microscopy (Leica DMI6000B) with a 5× objective for 60 min at 37°C. For quantification of the area under the curve (AUC; t = 60 min, baseline = 100%) a CellProfiler-based algorithm was used (Hagemeijer et al., in preparation). Experiments were performed in triplicate and repeated 3-4 times.
Statistical analyses. Statistical comparisons were made using one-way ANOVA followed by Dunnett’s or Tukey’s post-hoc tests for parametric data or using Kruskal-Wallis test followed by Dunn’s post-hoc test for nonparametric data (GraphPad Prism v.6.01). P values <0.05 were considered significant.