Cellsdirect kit

A549 cells were transfected according to manufacturer’s instructions (Dharmafect 1; Thermo Scientific) with ON-TARGET plus smart pool siRNA targeting RelA, human TLR5 or a non-targeting control 24 h prior to treating with flagellin and pp infection. Silencing was assessed by PCR/western blotting. RNA was prepared using the Qiagen RNeasy kit and amplified for TLR5 using human TLR5 primer pair Hs01019558_m1 (Thermo Scientific) in a quantitative reverse-transcription PCR (qRT-PCR) in accordance with the manufacturer’s guidelines (CellsDirect kit; Thermo Scientific) and fluorescence monitored in a 7500 real-time PCR machine (Applied Biosystems). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was included as an endogenous control for amplification efficiency, and TLR5 amplification normalized to GAPDH using the ΔΔCt method. Proteins were separated using an 8% SDS-PAGE gel, transferred to PVDF membrane (Millipore) and probed using a primary RelA (p65) rabbit monoclonal antibody (Cell Signaling Technology) or α-actin mouse monoclonal antibody (Sigma Aldrich) and secondary donkey α-rabbit HRP antibody (GE Healthcare) or sheep α-mouse HRP (GE Healthcare). Membranes were incubated in EZ-ECL prior to image capture using a PXi Multi-application gel imaging system (Syngene).

Amplification reactions were done using the CellsDirect kit (Thermo-Fisher) essentially according to manufacturer’s instructions, with the following modifications. The 31 gene multiplex primer set was added to individual lysates (100 nM final) in a final volume of 10 μL. Tubes were heated at 80°C for 10 min and chilled on ice for 3 min. 10 μL of 2x reaction buffer and 1 μL of SuperScript III/Platinum Taq (Thermo-Fisher) were added and tubes were reacted in a PCR machine at 50°C for one hour, followed by 85°C for 15 min to inactivate the reverse transcriptase. PCR amplification was then performed with an initial activation at 94°C for 2 min, followed by 18 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. After amplification, 20 μL of 10 mM Tris 7.5 was added to each sample to bring up the total volume to 40 μL. Four μL of each sample was then screened by quantitative PCR to determine expression levels of Gapdh (indicating successful capture and amplification) and Ncam1 (indicating an OSN). Taqman primers were designed to amplify regions internal to the 31 gene multiplex primer sequence, and samples were run on an ABI 7500 (Thermo-Fisher). Only cells with Ct values ≤ 25 for both genes were used for the NanoString analysis (Seattle, WA). See Figure 1—source data 1.