Anti nf κb p65 sc 372 or sc8008

For immunocytochemistry, U251 cells, U373 cells, and ITSCs treated as described above were fixed using 4 % paraformaldehyde (PFA, 4 % paraformaldehyde, 100 mM NaH 2 PO 4 , 0.4 mM CaCl 2 ) for 20 minutes followed by permeabilization and blocking with 0.02 % PBT (0.02 % TritonX-100 (Sigma-Aldrich cat. No. T8787, Taufkirchen, Germany) in PBS) comprising 5 % goat serum (Dianova, Hamburg, Germany). Primary antibodies anti-NF-κB-p65 (sc-372 or sc8008, Santa Cruz Biotechnologies) or anti-IRF3 (sc-9082, Santa Cruz Biotechnologies) were applied for 1 h. Respective secondary fluorochrome-conjugated antibodies (A21428, A21422, A21201, Molecular Probes, Göttingen, Germany) were subsequently added for 1 h under exclusion of light. Nuclear counterstaining was performed by applying 4′,6-Diamidin-2-phenylindol (DAPI; 1:2000, Life Technologies, Darmstadt, Germany) for 15 min followed by covering with mowiol. Fluorescence imaging was done using confocal scanning microscopy (LSM 780, Carl Zeiss, Jena, Germany) and ZEN software (Carl Zeiss). Image processing and analysis were performed using Fiji (31) followed by statistical analysis with GraphPad Prism software (GraphPad Software, La Jolla, CA, USA).

Nasal slices cultivated and treated as described above were removed from the culture inserts followed by preparation of 10 µm thick cryosections. For immunohistochemistry, 4 % PFA was applied for 20 minutes followed by permeabilization and blocking with 0.1 % PBT (Sigma-Aldrich) comprising 5 % goat serum (Dianova) and addition of primary antibodies anti-NF-κB-p65 (sc-372 or sc8008, Santa Cruz Biotechnologies), anti-IRF3 (sc-9082, Santa Cruz Biotechnologies) or anti-CK14 (sc-7156, Santa Cruz Biotechnologies) for 2 h. Respective secondary fluorochrome-conjugated antibodies (A21428, A21422, A21201, Molecular Probes) were subsequently applied for 1 h under exclusion of light. Nuclear counterstaining was done using DAPI (1:2000, Life Technologies) for 15 min followed by covering with mowiol. Fluorescence imaging was done via confocal scanning microscopy (LSM 780, Carl Zeiss) and ZEN software (Carl Zeiss) followed by image processing and analysis using Fiji (31) . Statistical analysis was performed using GraphPad Prism software (GraphPad Software).