Extended Data Fig. 1: Barrier function and polarized response by T84 cells.

T84 cells were seeded onto transwell inserts. (a). The rate of establishment of barrier function was measured by the trans- epithelial electrical resistance (TEER) using a chop stick electrode. TEER greater than 1000 Ohm/cm² indicates a tight barrier and is marked with a dashed line. Each point represents the mean +/- sd. n=6. (b). Barrier integrity was assessed by dextran diffusion assay. T84 cells were allowed to grow on transwells and at the indicated time points 4kDa fluorescent FITC-Dextran was added to the apical chamber. Three hours post-incubation, basolateral media was collected and analysed by fluorometry to assess the amount of dextran which has diffused to the basolateral compartment of the transwells. Results represent the mean +/- sd. n=3. n.d.= not determined. (c) pBMCs were isolated and treated with 10-fold serial dilutions of the indicated TLR agonists (starting concentrations: TLR1/2 (PAM3CSK4, 1 μg/mL); TLR3 (1:1 HMW and LMW poly(I:C), 10 μg/mL); TLR4 (LPS-EK, 1 μg/mL); TLR5 (Flagellin-ST, 1 μg/mL); TLR7/8 (R848, 1 μg/mL), TLR9 (ODN 2395 5 μM)). Supernatants were harvested 24 h post-stimulation and were tested for IL-6 by ELISA. Results represent the mean +/- sd. n=3. (d) Polarized T84 cells were fed FITC-labelled poly(I:C) in a side specific manner. Cells were harvested at indicated time points, were washed with a quick acid wash to remove any non-internalized poly(I:C) bound to the cell surface. Following acid wash, cells were lysed and the relative amount of internalized FITC-labelled poly(I:C) was measured by spectrofluorometry. Results represent the mean +/- sd. n=3. P=values were calculated from a two-tailed unpaired t test using GraphPad Prizm.