Peter van Mourik

44 Chapter 3 forskolin with or without potentiator(s) was added at concentrations as indicated and organoids were directly analyzed by confocal live cell microscopy (LSM710, Zeiss, 5× objective) for 60 min at 37 °C. Two wells were used per condition and experiments were repeated 2–5 times for F508del / S1251N (3 donors) and F508del / F508del (3 donors) organoids and 3–4 times for F508del / G551D organoids (1 donor). Quantification of forskolin-induced swelling Forskolin-stimulated organoid swelling was automatically quantified using Volocity imaging software (Improvision). The total organoid area (xy plane) increase relative to t = 0 of forskolin treatment was calculated. In some cases, cell debris and unviable structures were manually excluded based on criteria described in detail in a standard operating procedure (SOP). The area under the curve (AUC; t = 60; baseline = 100%) was calculated using Graphpad Prism. An unpaired T-test was used to calculate statistical differences (Fig. 2 - 4). Western blot analysis Organoids with S1251N or G551D from a 7 day-old culture were passaged 1:1 in 24- well plates and incubated with DMSO, VX-770, genistein, curcumin or combinations (1 well per condition) in 0.5 ml complete growth medium for 48 hours. The medium with compounds was refreshed after 24 h. Cells were lysed in Laemmli buffer supplemented with complete protease inhibitor tablets (1:50; Roche). Lysates were analyzed by SDS-PAGE and electrophoretically transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was blocked with 5% milk protein in TBST (0.3% Tween, 10 mM Tris pH8 and 150 mM NaCl in H 2 O) and probed 3 h at RT with mouse monoclonal E-cadherin-specific (1:10000; DB Biosciences) or CFTR- specific antibodies (450, 570 and 596; 1:5000; Cystic Fibrosis Folding consortium), followed by incubation with HRP-conjugated secondary antibodies (1:3000) and ECL development. An unpaired T-test was used to calculate statistical differences (Fig. 2k and 3j). RESULTS Intestinal current measurements of F508del / S1251N rectal biopsies treated with VX-770 and genistein Because different CFTR activation mechanisms for VX-770 (ATP-independent) and genistein (ATP-dependent) have been described 20,24-27 , we first assessed treatment of VX-770, genistein and their combination by intestinal current measurements (ICM) on human rectal biopsies derived from 17 F508del homozygotes or 7 compound heterozygotes expressing F508del and the gating mutation S1251N (Fig. 1). We