Peter van Mourik

50 Chapter 3 a b c d e f g Suboptimal [potentiator] (0.2 µ M forskolin) Near-saturating [potentiator] (0.03 µ M forskolin) 0.008 0.02 0.05 0.128 0.32 0.8 2 5 0 1000 2000 3000 4000 [Forskolin] ( µ M) DMSO Genistein (10 µ M ) VX-770 (0.1 µ M ) VX-770+genistein Organoidswelling (AUC t=60min) 0.008 0.02 0.05 0.128 0.32 0.8 2 5 0 1000 2000 3000 4000 [Forskolin] ( µ M) DMSO Genistein (50 µ M ) VX-770 (3 µ M ) VX-770+genistein Organoidswelling (AUC t= 60min) 0 3.125 6.25 12.5 25 50 100 200 0 700 1400 2100 2800 3500 [Curcumin] ( µ M) Fsk0.5 µ M Fsk5 µ M Organoidswelling (AUC t=60min) 0 3.125 6.25 12.5 25 50 100 200 0 700 1400 2100 2800 3500 [Genistein] ( µ M) Fsk0.5 µ M Fsk5 µ M Organoidswelling (AUC t=60min) 0 0.00003 0.0003 0.003 0.03 0.3 3 30 0 1000 2000 3000 4000 [VX-770] ( µ M) Fsk0.5 µ M Fsk5 µ M Organoidswelling (AUC t= 60min) Forskolin (0.128 µ M) t = 60 min t = 0 min DMSO Genistein (50 µ M) VX-770 (3 µ M) VX-770 + Genistein h CFTR E-cadherin C-band B-band F508del /G551D CFTRC-band (Normalized) j i F508del /G551D DMSO VX-770 (3 µ M ) Genistein (10 µ M ) Curcumin (10 µ M ) VX-770+gen VX-770+ cur Gen+ cur VX-770+gen+ cur Fig. 2g Fig. 2h 48 h VX-770 (3 µ M) Genistein (50 µ M) Curcumin (50 µ M) VX-770+gen VX-770+cur Gen+ cur VX-770+gen+cur 0 600 1200 1800 2400 Organoidswelling (AUC t=60min) VX-770 (0.1 µ M) Genistein (10 µ M) Curcumin (10 µ M) VX-770+gen VX-770+cur Gen+ cur VX-770+gen+cur 0 500 1000 1500 2000 Organoidswelling (AUC t=60min) p=0.0203 ns p=0.0016 ns p=0.0153 p=0.0188 p=0.0016 p=0.0384 p=0.0027 p=0.0143 p=0.0017 p=0.0018 Calculated additivevalues Measured values Calculated additivevalues Measured values 0 50 100 150 DMSO VX-770 (3 µ M ) Genistein (10 µ M ) Curcumin (10 µ M ) VX-770+gen VX-770+ cur Gen+ cur VX-770+gen+ cur 48 h p=0.0319 Figure 3. Incubation of VX-770, genistein and curcumin in rectal organoids derived from a F508del / G551D CF subject. (a-c) Forskolin(fsk)-induced swelling (FIS) expressed as the absolute area under the curve (AUC) calculated from time tracings shown in Fig. 2b (baseline = 100%, t = 60 min) of organoids stimulated with multiple dosages of VX-770 (a), genistein (b) or curcumin (c) at the indicated forskolin concentrations. (d) Representative confocal images of calcein-green-labeled organoids at the indicated time points of forskolin (0.128 μM) stimulation. Scale bar = 130 μm. (e,f) FIS of organoids stimulated with near-saturating (e) or suboptimal (f) concentrations of VX-770, genistein or both using a dose range of forskolin. (g,h) FIS of organoids stimulated with near-saturating (g) or suboptimal (h) concentrations of VX-770, genistein (gen), curcumin (cur) or their combinations at the indicated concentrations of forskolin, corrected for the FIS without addition of a potentiator. (a-c and e-h represent data from 1 F508del / G551D subject measured at 3 to 4 independent culture time points in duplicate. Mean ± SD. The SD indicates the inter-experiment variation). (i) Expression of CFTR and E-cadherin in whole cell lysates of F508del / G551D organoids detected by Western blot upon treatments as indicated (48 h). Immature (B-band) and mature (C-band) CFTR is indicated. (j) Quantification of CFTR C-band by Image J from Western Blots as shown in i. Data were normalised to the average of the 8 conditions per experiment. Data of 3 independent experiments was averaged. P value is indicated only for the condition that is statistically different from the DMSO control Mean ± SD.