Speedmill plus

Porcine skin biopsies were homogenized in QIAzol using innuSPEED Lysis tubes and the Speedmill Plus instrument (Analytik Jena GmbH, Jena, Germany). RNA from the homogenized tissue was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. NanoDrop One microvolume spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify RNA. For gene expression analysis, 1 µg of total RNA was reverse transcribed into single-stranded cDNA using the iScript™ Reverse Transcription Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Commercially available PrimePCR™ probe assays for the genes of interest and the housekeeping gene (Table 1) were purchased from Bio-Rad and the TaqMan™ Gene Expression Master Mix from Thermo Fisher Scientific. Real-time PCR was performed on a Bio-Rad CFX384 cycler. Relative target gene expression was normalized to the reference gene YWHAZ and calculated using the ΔΔCq method. All samples were calibrated with a blank skin sample. N-fold expression levels are shown as mean (bar) and standard deviation (whiskers). Cq values of duplicates deviating more than one cycle were excluded from further analysis.

For protein extraction, lung tissue samples were lysed in RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) with added inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), followed by homogenisation using the SpeedMill PLUS (Analytik Jena, Jena, Germany) and innuSPEED lysis Tube P (Analytik Jena). After centrifugation (5 min, 3000 rpm, 4 °C), supernatants were incubated on ice for 45 min, followed by centrifugation (1 × 5 min, 3000 rpm, 4 °C; 1 × 45 min, full speed, 4 °C). Finally, protein concentration was calculated after using Bradford Assay (Protein Assay Dye Reagent Concentration, Bio-Rad, Munich, Germany). Western blot analysis to detect Foxp3 and β-Actin (Table S6) was performed with 50 µg of total lung protein. Quantification of Foxp3/β-Actin protein levels was performed using the AlphaView Software for FluorChem Systems (Biozym Scientific, Oldendorf, Germany) as previously described.^{19 (link)}

Lung tissues of challenged mice were weighed and homogenized in 1 ml DMEM with 2% FBS and 1% P/S by SpeedMill PLUS (Analytik Jena AG, Jena, Germany) and clarified by centrifugation at 13,000 rpm for 10 min at 4°C. Infectious viral titers were determined by incubating 10-fold serially diluted samples with Vero-E6 cells in quadruplicate and cytopathic effects (CPEs) were observed after 4 days. Viral titer was expressed as 50% tissue culture infectious dose (TCID₅₀)/ml, which was calculated by the Reed and Muench method.