7500 software version 2

After optimization, primers and probes were ready for PCR amplification. One-step Real-time PCR was performed in a 20μL reaction mixture after optimization. It consisted of 10μL 2x One Step PCR Mix (Vazyme), 1μL Enzyme Mix (Vazyme) containing reverse transcription enzyme and DNA polymerase, 0.35μL H5-HA probe (20μM), 0.35μL NP probe (20μM), 0.4μL H5-HA reverse primer (20μM), 0.4μL H5-HA forward primer (20μM), 0.4μL NP reverse primer (20μM), 0.4μL NP forward primer (20μM), 6μL RNA sample, 0.4μL 50x ROX and 0.3μL RNase-free water. Reactions were carried through in ABI 7500 Real-time PCR instrument (Applied Biosystems) with the following programs: 15min at 55°C, 5min at 95°C, 40 cycles of 5s at 95°C and 34s at 60°C. Meanwhile, NTC (no template control) and positive control (RNA from H5 subtype AIV) were both used. The data was then analyzed with 7500 Software Version 2.0.6 (Applied Biosystems).

Total RNA from treated HUVEC was isolated using High Pure RNA Isolation Kit (Roche Diagnostics, Mannheim, Germany). RNA integrity has been analyzed by Agilent 2100 bioanalyzer using Eukaryote Total Nano 2.6 assay. Reverse transcription of mRNA into cDNA was performed with SuperScript II Reverse Transcriptase according to manufacturer's instructions (Thermo Fisher Scientific, Waltham, MA, USA) using 500 ng of total RNA and random hexamer primers. Quantification was performed by real-time PCR (7500 Fast Real-Time PCR System, Thermo Fisher Scientific, Waltham, MA, USA) using GoTaq qPCR Master Mix (Promega, Mannheim, Germany) with specific primers (Sigma-Aldrich, Munich, Germany/for primer sequences see Table 1). POLR2A was used as reference gene for cDNA content normalization. Amplification started with an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, specific annealing and extension for each gene for 60 s. Melt‐curve analysis was performed following every run to ensure a single amplified product in each reaction. Analysis of the raw data was performed with the 7500 Software Version 2.06 (Applied Biosystems by Life Technologies, Darmstadt, Germany). Data were evaluated using a mathematical model of relative expression ratio in real-time PCR under constant reference gene expression [43] (link).

Total RNA was measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific), and a reverse-transcription reaction was performed with the iScriptTM kit (Bio-Rad, Hercules, CA, USA). For real-time polymerase chain reaction, complementary DNA was incubated with SYBR Green (Applied Biosystem, Waltham, MA, USA) and the primers for all target genes. All primer sequences for NLRP1, NLRP3, NLRP4, IL-18, IL-1B and CARD8 were synthesized by Invitrogen, and the sequences are listed in Supplementary Materials Table S1. The DNA amplification was carried out in a 7500 real-time PCR system (Applied Biosystems, Waltham, MA, USA), and data analysis was performed using the 7500 software, version 2.0.6 (Applied Biosystems), according to the ΔΔ-cycle threshold method [51 (link)].

From 10 µm FFPE sections total RNA isolation and DNAse digestion were performed, using a standardized fully automated isolation method based on germanium-coated magnetic beads (XTRAKT RNA kits, STRATIFYER Molecular Pathology GmbH, Cologne, Germany) in combination with the liquid handling robot XTRAKT SL (STRATIFYER) as previously described [36] (link) for the analysis of TNF, IL10, TGFβ1 and CALM2 (reference gene). For the analysis of IL4, IL13, GM-CSF, M-CSF and CALM2 (reference gene) RNA was isolated using Maxwell 16 LEV RNA FFPE Kit, according to the manufacturer's instructions. A probe of L1236-RNA was used as positive control and for comparison of the PCR-runs. Standard and no-template controls were included. ΔΔC_T qRT-PCR with probes for CALM2 (reference gene), TNF, IL10 and TGFβ1 as well as for IL4, IL13, GM-CSF and M-CSF was performed with 40 cycles of nucleic acid amplification using QuantiFast Probe Assays for one-step qRT-PCR (Qiagen, Hilden, Germany). Assays were analyzed by the Applied Biosystems 7500 Fast Real-Time PCR System and the 7500 Software Version 2.0.6 (Applied Biosystems, Darmstadt, Germany).

Tissue was lysed with RNA lysis buffer and homogenized with a Precellys homogenizer (Peqlab Biotechnologie GmbH, Erlangen, Germany). Total RNA was isolated using a peqGold Total RNA Kit (Peqlab Biotechnologie GmbH, Erlangen, Germany) and determined using a NanoDropTM 1000 Spectrophotometer (Peqlab Biotechnologie GmbH, Erlangen, Germany). Reverse transcription was performed with SuperScript II Reverse Transcriptase according to the manufacturer’s instructions (Life Technologies, Darmstadt, Germany). The mRNA expression was determined by SYBR green-based real-time PCR reactions using specific primers. Quantification was performed by real-time PCR with GoTaq qPCR Master Mix (Promega, Mannheim, Germany). Analysis of raw data was performed with 7500 Software version 2.06 (Applied Biosystems by Life Technologies, Darmstadt, Germany). Evaluation of the data was carried out using a mathematical model of relative expression ratio in real-time PCR under constant reference gene expression [27 (link)]. The primers used are listed in Table 2.