Analysis of relative expression of iNOS mRNA (A) and nitric oxide (NO) production (B) in PAM pre-treated with T-2 toxin. A- T-2 toxin affects iNOS mRNA expression: Relative mRNA expression of iNOS in PAM treated or not by 3 nM of T-2 toxin for 1 h before their activation with each specific TLR-agonist during 4 h. Data were analyzed with DART-PCR software and iNOS expression was normalized by the mean of two housekeeping genes (β2-microglobuline and RPL32). Gene expression of iNOS was reported to unstimulated PAM whose expression is visualized by the line level at 1. Significant differences (P < 0.05) between unstimulated PAM (negative control) and cells treated (with TLR-agonists in the absence or presence of T-2) are marked with an asterisk. B- T-2 toxin affects NO production: the cells were cultured for 1 h in the presence (black bars) or absence (white bars) of 3 nM of T-2 toxin before their activation with different TLR-agonists: TLR2-agonist (HKLM); TLR4-agonist (LPS); TLR2/6-agonist (FSL1) and TLR7-agonist (Imiquimod) for 16 h. An incubation of both cells and DAF-FM probe was used to determine the production of NO. Mean Fluorescence Intensity (MFI) of DAF-FM +/− SEM was determined by flow cytometry analysis and then normalized with the negative control. Mean (+/− SEM) of values obtained with PAM from five different piglets are shown. * indicates a P value < 0.05.