The effects of SFN on LPS induced NF-κB and TBP expression.

<p>moDCs were pre-incubated for 1 h with or without SFN (10 μM) before exposure to LPS (1.0 μg/ml) for 0, 1, 3, 6, 12, 24 h or to the indicated time. The transcription factor NF-κB and TBP mRNA expression were quantified by qRT-PCR (A and C). Data are mean ± standard deviations (SD) (* <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001) of triplication samples from three independent experiments. NF-κB and TBP protein expression were examined by western blotting. The results were determined from one experiment representative of two experiments. The p50 and p65 protein of NF-κB family were analyzed by western blotting by the selected time points (0, 3, 12, and 24 h) (B). The western blotting result was from one experiment of three independent experiments.</p

SFN pre-incubation inhibited LPS induce cell death in a time denpendent manner.

<p>moDCs at day 7 were used for cell viability assay by WST-1 kit. A SFN dose-dependent assay was used to confirm cell viability of moDCs after stimulating with different concentration of SFN (Control, 5 μM, 10 μM, 15 μM, 20 μM, and 50 μM) for 24h (A). For the effects of SFN on LPS induced cell death, moDCs were pre-incubated 24 h with or without SFN (10 μM) before exposed to LPS (1 μg/ml) for 1, 3, 6, 12, and 24 h (B). The results were combined from three independent experiments and each experiment was performed in triplicate. The data were represented as the mean ± standard deviations (SD) (* <i>P</i> < 0.05; **<i>P<</i>0,01; ***<i>P<</i>0,001).</p

The effects of SFN on LPS induced up-regulation of TLR4 and MD2 gene expression.

<p>moDCs were pre-incubated for 1 h with or without SFN (10 μM) before stimulation for 0, 1, 3, 6, 12, 24 h with LPS (1.0 μg/ml). The TLR4 and MD2 mRNA expression were quantified by qRT-PCR. The results were combined from three independent experiments and each experiment was performed in four replications. The data represented as the mean ± standard deviations (SD) (* <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001).</p

SFN affects gene expressions and protein productions of cytokine.

<p>moDCs were pre-incubated for 24 h with or without SFN (10 μM) before stimulation with LPS (1 μg/ml) for additional 24 h. The cell lysate proteins of IRF6 and TNF-α were analyzed using western blotting (A). The effects of SNF on gene expression of pro-inflammatory cytokines TNF-α, IL-1ß, IL-8, and IFN-γ were quantified by qRT-PCR (B). The pro-inflammatory cytokines TNF-α (C) and IL-1ß (D) secreted in cell culture supernatant were determined by ELISA. The results were combined from three independent experiments and each experiment was performed in triplicates. The data were represented as the mean ± standard deviations (SD) (* <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001).</p

Expression of class I, class II, and class IV HDAC genes in porcine moDCs stimulated with LPS.

<p>The expression of HDAC family genes in moDCs were influenced with LPS (1μg/ml) stimulation for 24 h. moDCs were generated from adherent monocytes at day 7 <i>in vitro</i>, which were treated with or without LPS. The class I (A), class II (B), and class IV (C) of HDACs mRNA were quantified by qRT-PCR and normalized with the housekeeping gene HRPT1. The results were combined from three independent experiments and each experiment performed in triplicate. The data were represented as the mean ± standard deviations (SD) (* <i>P</i> < 0.05; ** <i>P</i> < 0.01).</p

The effects of LPS on DNMT gene expression.

<p>Expression of genes that encode the enzymes responsible for methylating CpG sites of DNA were quantified by qRT-PCR including the maintenance methyltransferase DNMT1 and the <i>de novo</i> methyltransferase DNMT3a in moDCs in response to LPS exposure (24 h) compared with control. The results were combined from three independent experiments and each experiment was performed in triplicate. The data were represented as the mean ± standard deviations (SD) (* <i>P</i> < 0.05; ** <i>P</i> < 0.01).</p

Biological process GOs of Duroc cluster 32 post PRRSV infection.

<p>Biological process GOs of Duroc cluster 32 post PRRSV infection.</p

Top 10 scored pathways using KEGG and clustered RNA-Seq dataset post PRRSV infection, represented by the cluster ID of Duroc lung DCs.

<p>Top 10 scored pathways using KEGG and clustered RNA-Seq dataset post PRRSV infection, represented by the cluster ID of Duroc lung DCs.</p

SFN inhibits LPS induced moDC maturation and enhances the phagocytic activity.

<p>moDCs at day 7 in culture were used for cell phagocytosis and cell differentiation status analysis. moDCs were pre-incubated for 1 h with or without SFN (10 μM) before stimulation for 24 h LPS (1.0 μg/ml) or to the indicated concentrations. CD40, CD80, and CD86 cellular surface markers expression were analyzed by flow cytometry (A). The flow cytometry results shown were from one experiment of two independent experiments. CD40, CD80 and CD86 mean fluorescence intensity (MFI) determined by flow cytometry (B). The flow cytometry results were combined from two independent experiments and each experiment was performed from triplications. Data are mean ± standard deviations (SD) (the letters a and b <i>P<</i>0.01). The phagocytic activity of moDCs was examined after stimulating with different concentration of LPS (0,5 μg/ml, 1,0 μg/ml, and 2,0 μg/ml) with or without 24 h pre-treatment with SFN (C). The mRNA expression of DCs surface markers CD40, CD80 and CD86 were quantified using qRT-PCR (D). The mRNA expression and phagocytosis results were combined from three independent experiments and each experiment was performed in four replications. The data represented as the mean ± standard deviations (SD) (* <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001).</p

List of primer sequences used in this study.

<p>F: Forward primer; R: Reverse primer; bp: base pair.</p><p>List of primer sequences used in this study.</p