Peter van Mourik

48 Chapter 3 0 µ M 0.3 µ M 3 µ M [VX-770] a b c g h d e f i Forskolin ( 0.128 µ M) t= 60min t= 0min AUC t= 60min 0 0.00003 0.0003 0.003 0.03 0.3 3 30 0 700 1400 2100 2800 3500 [VX-770] ( µ M) Fsk0.128 µ M Fsk0.5 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) Fsk0.128 µ M Fsk0.5 µ M Organoidswelling (AUC t=60min) 0 3.125 6.25 12.5 25 50 100 200 0 700 1400 2100 2800 3500 [Curcumin] ( µ M) Fsk0.128 µ M Fsk0.5 µ M 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) 0.008 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) Suboptimal [potentiator] (0.08 µ M forskolin) Near-saturating [potentiator] (0.008 µ M forskolin) 0 10 20 30 40 50 60 100 120 140 160 180 Time (min) Normalizedorganoidarea (%) 0 0.00003 0.0003 0.003 0.03 0.3 3 30 [VX-770] ( µ M): 0.128 µ M fsk Fig. 2h Fig. 2i C-band B-band F508del /S1251N 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 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 F508del /S1251N CFTR E-cadherin j k Figure 2. Incubation of VX-770, genistein and curcumin in rectal organoids derived from F508del / S1251N CF subjects. (a) Representative confocal images of calcein-green-la- beled F508del / S1251N organoids at the indicated time points of forskolin (0.128 μM) stimulation. Scale bar = 130 μm. (b)A time tracing of the forskolin(0.128 μM)-induced surface area increase relative to t = 0 (normalized area) of F508del / S1251N organoids at different VX-770 concentrations averaged from two independent wells. Mean ± SD. (c-e) Forskolin(fsk)-in- duced swelling (FIS) expressed as the absolute area under the curve (AUC) calculated from time tracings shown in b (baseline = 100%, t = 60 min) of organoids stimulated with multiple dosages of VX-770 (c), genistein (d) or curcumin (e) at the indicated forskolin concentrations. (f,g) FIS of organoids stimulated with near-saturating (f) or suboptimal (g) concentrations of VX-770, genistein or both using a dose range of forskolin. (h,i) FIS of organoids stimulated with near-saturating (h) or suboptimal (i) 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. (c-i represent data averaged from 3 F508del / S1251N sub- jects. Each subject was measured at 2 to 5 independent culture time points in duplicate. Mean ± SD.The SD indicates the inter-subject variation). (j) Expression of CFTR and E-cadherin inwholecell lysatesofF508del /S1251NorganoidsdetectedbyWesternblotupon treatmentsas indicated (48h). Immature (B-band)andmature (C-band)CFTR is indicated. (h)Quantifi- cationofCFTRC-bandby ImageJ fromWesternBlotsasshown in j.Datawerenormalised to theaverageof the8conditionsperexperiment.Dataof3differentF508del /S1251Ncultures were averaged, each culture was assessed in 1-3 independent experiments. P values are indicated only for the conditions that were statistically different from the DMSO control. Mean ± SD. 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 0 500 1000 1500 2000 Organoidswelling (AUC t=60min) p=0.0193 p=0.0306 p=0.0004 ns p=0.0285 p=0.0142 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 0 600 1200 1800 2400 Organoidswelling (AUC t=60min) Calculatedadditivevalues Measuredvalues p=0.0196 ns p=0.0440 ns ns ns Calculated additivevalues Measured values 0 50 100 150 p=0.0241 48 h CFTRC-band (Normalized) Figure 2. Incubation of VX-770, genistein and curcumin in rectal organoids derived from F508del / S1251N CF subjects. (a) Representative confocal images of calcein-green-labeled F508del / S1251N organoids at the indicated time p ints of f rskolin (0.128 μM) stimulation. Scal b r = 130 μm. (b) A time tracing of the forskolin(0.128 μM)-induced surface area increase relative to t = 0 (normalized area) of F508del / S1251N organoids at different VX-770 concentrations averaged from two independent wells. Mean ± SD. (c-e) Forskolin(fsk)-induced swelling (FIS) expressed as the absolute area under the curve (AUC) calculated from time tracings shown in b (baseline = 100%, t = 60 min) of organoids stimulated with multiple dosages of VX-770 (c), genistein (d) or curcumin (e) at the indicated forskolin concentrations. (f,g) FIS of organoids stimulated with near-saturating (f) or suboptimal (g) concentrations of VX-770, genistein or both using a dose range of forskolin. (h,i) FIS of organoids stimulated with near-saturating (h) or suboptimal (i) 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. (c-i represent data averaged from 3 F508del / S1251N subjects. Each subject was measured at 2 to 5 independent culture time points in duplicate. Mean ± SD. The SD indicates the inter-subject variation). (j) Expression of CFTR and E-cadherin in whole cell lysates of F508del / S1251N organoids detected by Western blot upon treatments as indicated (48 h). Immature (B-band) and mature (C-band) CFTR is indicated. (h) Quantification of CFTR C-band by Image J from Western Blots as shown in j. Data were normalised to the average of the 8 conditions per experiment. Data of 3 different F508del / S1251N cultures were averaged, each culture was assessed in 1-3 independent experiments. P values are indicated only for the conditions that were statistically different from the DMSO control. Mean ± SD.