<p>(<b>A</b>) The expression of <i>Dcaf7</i>, <i>Dusp3</i>, and <i>Psme3</i> in A/J was significantly lower than C57BL/6J under both non-infected and <i>S. aureus</i> infected conditions. Eight-week-old male A/J and C57BL/6J mice (n = 6) were challenged (i.p.) by <i>S. aureus</i> Sanger 476 at 10<sup>7</sup> CFU/g or DPBS. At two hours post-infection whole blood RNAs was extracted by RNeasy followed by RT-PCR and qPCR. <i>Dcaf7</i> (0.81 fold; p<0.05), <i>Dusp3</i> (0.27 fold; p<0.01), and <i>Psme3</i> (0.83 fold; p<0.05) were down-regulated in A/J mice at baseline (0 hr) as compared with resistant C57BL/6J. Two genes exhibited elevated expression in susceptible A/J mice (baseline): <i>Fam134c</i> (1.82 fold; p<0.01) and <i>Slc4a1</i> (1.31fold; p<0.05). The baseline difference in expression between susceptible (A/J) and resistant (C57BL/6J) mice of all the five genes remained unchanged at 2 hr post <i>S. aureus</i>-infection. (<b>B</b>) <i>Dusp3</i> and <i>Psme3</i> inhibit NF-κB signaling activity in RAW264.7 macrophages. RAW264.7 cells co-transfected with NF-κB-luciferase and pRL-TK plasmids were then transfected with siRNA of each individual candidate genes (<i>Dcaf7</i>, <i>Dusp3</i>, <i>Psme3</i>) or scrambled siRNA. Then transfected RAW cells were stimulated by either medium alone, medium containing LTA (10 µg/ml) or medium containing <i>S. aureus</i> particles (10 µg/ml) for 7 hours. Cells were directly lysed by 1× passive lysis buffer and luciferase activity was assayed and normalized to renilla activity as previously described <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004149#ppat.1004149-Yan1" target="_blank">[15]</a>. As shown, knockdown of both <i>Dusp3</i> and <i>Psme3</i> significantly up-regulates NF-κB luciferase activity (p<0.01). (<b>C</b>) Knockdown of <i>Dusp3</i> or <i>Psme3</i> enhanced the activation of NF-κB signaling upon <i>S. aureus</i> stimulation. BMDMs from C57BL/6J were transfected with either scrambled, <i>Dusp3</i> or <i>Psme3</i> siRNA, then stimulated with <i>S. aureus</i> for 15 minutes. Whole cell lysate was loaded for western-blot. Knockdown of <i>Dusp3</i> or <i>Psme3</i> dramatically increased degradation of IκBα and phosphorylation of p65 (Ser536) as compared with scrambled siRNA control. (<b>D</b>) Enhanced NF-κB signaling upon <i>S. aureus</i> stimulation in CSS11 BMDMs. BMDMs from either C57BL/6J or CSS11 were stimulated with <i>S. aureus</i> for 15 minutes. Whole cell lysate was loaded for Western blot. BMDMs from CSS11 exhibited increased degradation of IκBα and phosphorylation of p65 (Ser536) as compared with BMDMs from C57BL/6J. (<b>E</b>) Bay inhibition of NF-κB dramatically suppressed cytokine production. RAW264.7 macrophages were pre-treated with 4 µM Bay 11-7085 for one hour, then stimulated with 10 µg/ml <i>S. aureus</i> particles in 2 µM Bay for 3 hours. RNA was extracted and subjected to reverse-transcription PCR and qPCR. Inhibition of NF-κB by Bay inhibitor dramatically suppressed cytokine production upon <i>S. aureus</i> stimulation, including IL-1β (p<0.01), IL-6 (p<0.05) and TNF-α (p<0.05). (<b>F</b>) Inhibition of NF-κB enhanced <i>Dusp3</i> and <i>Psme3</i> expression. The inhibition of NF-κB activity by Bay inhibitor significantly enhanced the expression of both <i>Dusp3</i> (p<0.05) and <i>Psme3</i> (p<0.05), which indicated a reciprocal relationship between NF-κB signaling activity and <i>Dusp3</i> or <i>Psme3</i> expression.</p
