Inh 172

Roscovitine and M3 were synthesized at ManRos Therapeutics and used at concentrations ranging from 0.1 to 100 μM. TRPC6 inhibitor^{17 (link)} was provided by Dr. Stephanie Häfner and used at 10 µM. CFTR modulators tezacaftor and ivacaftor (Selleck chem) were used at 5 µM. Cysteamine (Sigma) was used at 5 µg/ml and the CFTR inhibitor Inh-172 (Sigma) was used at 5–10 µM. All reagents were dissolved in DMSO and therefore non-treatment conditions received equal DMSO concentrations. All reagents were given as pre-treatment for 6 h prior to general infection experiments, and 4 h prior to halide efflux and phagocytosis.

Cellular chloride secretion was assessed using a colorimetric assay that measures cellular iodide efflux as a surrogate for monitoring chloride ion channel activity, as per Tang and Wildey (2004) (link). Caco-2 cells were seeded in 96-well plates at 2.5 × 10⁴ cells/well and incubated for 24 h at 37°C. Cells were then loaded with 100 µl pre-warmed iodine loading buffer and incubated for 4 h at 37°C. Following three washes with sterile PBS, and incubated for 30 min with plantain NSP (2.5–10 mg/ml) or chloride ion channel agonists, forskolin (200 μM; Sigma) or RP107 (100 μM; Sigma) as a positive controls (Noel et al., 2006 (link)). Following incubation, cells were lysed with 1% (v/v) Triton X-100 in deionised water. Intracellular iodine concentration was determined using the modified Sandell-Kolthoff reaction, and absorbances measured at OD_410nm (Tang and Wildey, 2004 (link)). Chloride channel blockers, cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor Inh-172 (12.5–200 μM; Sigma) (Linsdell, 2014 (link)), or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 50–800 μM; Sigma) (Keeling et al., 1991 (link)), were evaluated for their ability to block cellular chloride channel activation, and were present during the 4 h iodine-loading step prior to incubation with soluble NSP from plantain, RP107 or forskolin.