Cftrinh 172

CFTR_inh-172 was purchased from Cayman Chemical (Ann Arbor, MI), 4,4’-Diisothiocyanatostilbene-2,2’-disulfonate (DIDS) and S3226 were purchased from Sigma-Aldrich (St. Louis, MO). DRA_inh-A250 and DRA_inh-A270 were gifts from Drs. Alan Verkman and Onur Cil at the University of California, San Francisco. Linaclotide was purchased from Santa Cruz Biotechnology, Inc (Dallas, TX).

For actomyosin inhibition, blebbistatin (Selleck Chemical) and Y-27632 (Selleck Chemical) were used at 100 μM concentration and added 1h before stimulation. For aquaporin 3 (AQP3) inhibition, copper sulfate (CuSO4, Sigma Aldrich) was used at 12.5μM concentration and added 24h before stimulation. For Na+/K+ ATPase inhibition, ouabain octahydrate (Sigma Aldrich) was used at 100nM and added 24h before stimulation. For CFTR channel inhibition, CFTR(inh)-172 (Cayman) was used at 50μM and 100μM concentration and added 24h before stimulation for MDCK cysts and 48h before stimulation for ISC organoids. For NKCC inhibition, bumetanide was used at 100μM concentration and added 1h before stimulation. For PI3K inhibition, LY294002 (Medchemexpress) was used at 25 μM to stall electrotaxis at and 50 μM to reverse electrotaxis for both cysts and monolayers. The LY294002 treated samples were incubated at 37 °C for 60 min before being assembled into the bioreactor. Media with the same concentration of LY294002 was used for perfusion. Inhibitors were applied after the patterning stencils were removed to prevent the inhibitors from being absorbed into the PDMS. Identical concentration of inhibitors was supplemented in the perfusion media to prevent the inhibitors from washing out during stimulation.