Multimode plate reader

Paracellular permeability was quantified by inulin-FITC (0.1 mg.mL⁻¹ in PBS or medium) perfusion of bioengineered intestinal tubules. This was done as described before [11 (link)]. In short, bioengineered intestinal tubules were washed with PBS and perfused with inulin-FITC for 10 min at a speed rate of 0.1 mL/min. Samples were taken from the chamber to assess inulin-FITC leakage by absorbance at excitation wavelength of 480–492 nm and emission wavelength of 518–520 nm using a multimode plate reader (Promega, Leiden, The Netherlands). Results are shown relative to positive control, unexposed bioengineered intestinal tubules. Bioengineered intestinal tubules were extracted from the 3D-chamber and cut in pieces and used for cell viability and alkaline phosphatase activity.

The culture supernatant sample or purified S protein was coated onto each well of the microtiter plate and left overnight at 4°C. After the wells were blocked with 1% BSA for 1 h, dilutions of human anti-S1 antibody (Abcam) were added to the wells and incubated for 3 h at room temperature. After being washed, HRP-conjugated rabbit anti-human IgG antibody (Abcam) was added and incubated for 1 h at room temperature. The bound antibodies were detected using TMB substrate (Sigma-Aldrich). Absorbance was read at 450 nm using a Multimode Plate Reader (Promega, Madison, WI, USA).

To demonstrate the direct interaction between miR-146a-5p and TRAF6, the 3′ UTR sequence of TRAF6 mRNA containing the putative miR-146a-5p binding sites was synthesized by Sangon Biotech (Shanghai, China), and sub-cloned to downstream of the firefly reporter gene in the pmirGLO vector (Promega, Madison, WI, United States), which was named pmirGLO-TRAF6-WT. Mutant TRAF6 3′ UTR, containing a mutated binding site for miR-146a-5p, was cloned into pmiRGLO and named pmiRGLO-TRAF6-MUT. The oligonucleotide sequences are listed in Supplementary Table S1. For the dual luciferase reporter assay, cells were cultured in a 96-well plate and co-transfected with 100 nM miR-146a-5p mimics and 200 ng pmirGLO-TRAF6-WT reporter plasmid or pmiRGLO-TRAF6-MUT vector using Lipofectamine 3000 (Invitrogen). The luciferase activity was measured 48 h after transfection, using the Dual-Glo Luciferase Assay System (Promega) and assayed with Multimode Plate Reader (Coring, NY, United States). The relative transcriptional activities were expressed as the fold change above the vehicle control in luciferase activity after normalization to renilla luciferase activity.