Peter van Mourik

47 Potentiator synergy in rectal organoids triple combination treatment, but not for the VX-770 + genistein condition (Fig. 2i) . In line with Fig. 2h and 2i, the response of VX-770- or genistein-treated organoids was dose-dependently increased by curcumin and appeared more prominent at higher curcumin concentrations (>12.5 μM; Supplementary Fig. S1b,c). Potentiator- induced swelling was absent in organoids expressing two class I CFTR mutations, indicating that the FIS induced by VX-770, genistein and curcumin is fully dependent on function of CFTR (Supplementary Fig. S2). We also assessed CFTR-S1251N protein expression detected by Western blot upon chronic stimulation (48 h) of VX-770, genistein, curcumin or combinations (Fig. 2j,k). Results indicated that the VX-770 + curcumin and triple combination treatments reduced expression of maturated CFTR C-band to ~70% of mock-treated organoids (Fig. 2k). In conclusion, all potentiator combinations synergistically repaired CFTR- dependent epithelial fluid transport in organoids with CFTR-S1251N, suggesting that VX-770, genistein and curcumin potentiate CFTR by different mechanisms. The synergy was detected at suboptimal activation of CFTR and suboptimal potentiator levels, conditions that may predominate in vivo . 3