Nf κb inhibitor

TMZ was dissolved in DMSO (35 mM stock) and stored in aliquots at −80°C. The cyclo-RGD peptide (BML- AM100-0001, Enzo Life Sciences) was diluted in PBS. The proteasomal inhibitor MG132 and the NFκB inhibitor (Merck Millipore) were dissolved in DMSO (10 mM and 30.4 mM stock, respectively), stored in aliquots at −20°C and applied at 10 μM.

Confluent Calu-3 cells grown in 24-well plates were washed once and then stimulated with 100–200 ng/ml standard flagellin isolated from Salmonella typhimurium (InvivoGen), 5 μg/ml Pam3Cys (InvivoGen), or 1 μg/ml LPS (Sigma-Aldrich) diluted in the infection medium for 24 h at 37°C. Subsequently, the cells were washed with PBS. Total RNA was harvested as described above and subjected to RT–qPCR. Treatment with 10 μM ERK inhibitor U0126 monoethanolate (U120; Merck), p38 MAPK inhibitor SB 202190 (S7067; Merck), or NF-κB inhibitor BAY 11-7821 (Merck Millipore) was performed for 1 h before flagellin stimulation. To analyze the expression of the TMPRSS2 protein, cells were treated with flagellin (200 ng/μl) for 24 h or remained untreated. Subsequently, cells were subjected to SDS–PAGE and Western blot analysis as described below.
Primary HBEC cultivated in 24-well plates were treated with flagellin diluted in the ALI-Mix medium (1:1 mixture of DMEM (Sigma-Aldrich) and AEGM, supplemented with 60 ng/ml retinoic acid) at the indicated concentrations for 4 or 24 h.