1. Potentiator synergy in rectal organoids carrying S1251N, G551D, or F508del CFTR mutations 
Johanna F. Dekkers, Peter Van Mourik, Annelotte M. Vonk, Evelien Kruisselbrink, Gitte Berkers, Karin M. de Winter-de Groot, Hettie M. Janssens, Inez Bronsveld, Cornelis K. van der Ent, Hugo R. de Jonge, Jeffrey M. Beekman 
Journal of Cystic Fibrosis 
Volume 15, Issue 5, Pages (September 2016)
DOI: /j.jcf
Copyright © 2016 European Cystic Fibrosis Society. Terms and Conditions

2. Fig. 1
Intestinal current measurements of F508del/S1251N rectal biopsies treated with VX-770 and genistein. (a) Examples of intestinal current measurement (ICM) tracings of an F508del/F508del or F508del/S1251N rectal biopsy incubated with compounds as indicated (amiloride 100 μM; forskolin (fsk) 10/100μM; VX μM; genistein 50μM; carbachol 100 μM). (b) Delta Isc ICM values of biopsies derived from 17 F508del/F508del (2–4 biopsies per subject) and 7 F508del/S1251N subjects (1–4 biopsies per subject).
Journal of Cystic Fibrosis  , DOI: ( /j.jcf )
Copyright © 2016 European Cystic Fibrosis Society. Terms and Conditions

3. Fig. 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 points of forskolin (0.128μM) stimulation. Scale bar=130μm. (b) A time tracing of the forskolin(0.128μM)-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=60min) 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. (Panels 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 (48h). 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 normalized 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.
Journal of Cystic Fibrosis  , DOI: ( /j.jcf )
Copyright © 2016 European Cystic Fibrosis Society. Terms and Conditions

4. Fig. 3
Incubation of VX-770, genistein and curcumin in rectal organoids derived from an 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=60min) 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 (0.128μM) stimulation. Scale bar=130μm. (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 (48h). 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 normalized 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.
Journal of Cystic Fibrosis  , DOI: ( /j.jcf )
Copyright © 2016 European Cystic Fibrosis Society. Terms and Conditions

5. Fig. 4
Incubation of VX-770, genistein and curcumin in rectal organoids derived from F508del/F508del CF subjects. (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=60min) of organoids stimulated with multiple dosages of VX-770 (a), genistein (b) or curcumin (c) at the indicated forskolin concentrations. (d, e) FIS of organoids stimulated with near-saturating (d) or suboptimal (e) concentrations of VX-770, genistein or both using a dose range of forskolin. (f) The effect of VX-809 preincubation (24h) on FIS of organoids stimulated with VX-770 or genistein using a dose range of forskolin. (g, h) FIS of organoids pre-incubated with VX-809 for 24h, and 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 VX-809-repaired FIS without addition of a potentiator. (All figures represent data averaged from 3 F508del/F508del subjects. Each subject was measured at 2 to 5 independent culture time points in duplicate. Mean±SD. The SD indicates the inter-subject variation.)
Journal of Cystic Fibrosis  , DOI: ( /j.jcf )
Copyright © 2016 European Cystic Fibrosis Society. Terms and Conditions

6. Supplementary Fig. S1
Dose-dependent potentiator synergy in F508del/S1251N organoids. (a–c) Forskolin-induced swelling of F508del/S1251N organoids stimulated with a dose range of genistein with or without VX-770 treatment (a), or stimulated with a dose range of curcumin with or without genistein (b) or VX-770 (c) at the indicated concentration of forskolin (this concentration was selected using Fig. 2f). The calculated additive responses of the single treatments are indicated as a dashed line. (Panels a–c 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.)
Journal of Cystic Fibrosis  , DOI: ( /j.jcf )
Copyright © 2016 European Cystic Fibrosis Society. Terms and Conditions

7. Supplementary Fig. S2
Incubation of VX-770, genistein and curcumin in rectal organoids derived from a CF subject expressing two class I CFTR mutations. Forskolin-induced swelling (FIS) of organoids stimulated with near-saturating concentrations of VX-770, genistein (gen), curcumin (cur) or combinations at a saturating concentration of forskolin, corrected for the FIS without addition of a potentiator. The subject was measured at 2 independent culture time points in duplicate. Mean±SD.
Journal of Cystic Fibrosis  , DOI: ( /j.jcf )
Copyright © 2016 European Cystic Fibrosis Society. Terms and Conditions

8. Supplementary Fig. S3
Dose-dependent potentiator synergy in F508del/F508del organoids. (a, b) Forskolin-induced swelling of F508del homozygous organoids stimulated with a dose range of genistein with or without VX-770 (a) or VX-770+VX-809 (b) at the indicated concentrations of forskolin. The calculated additive responses of the single treatments are indicated as a dashed line. (a and b represent data averaged from 3 F508del/F508del subjects. Each subject was measured at 2 independent culture time points in duplicate. Mean±SD. The SD indicates the inter-subject variation.) (c) Expression of CFTR and E-cadherin in whole cell lysates of F508del/F508del (2 different donors) and F508del/S1251N organoids detected by Western blot upon treatments as indicated (48h). Of note, higher compound concentrations were used as compared to Figs. 2j,k and 3i,j. Immature (B-band) and mature (C-band) CFTR is indicated.
Journal of Cystic Fibrosis  , DOI: ( /j.jcf )
Copyright © 2016 European Cystic Fibrosis Society. Terms and Conditions