Quantitative-PCR confirmed elevation of cytokine production in macrophages transfected by <i>Dusp3</i> and <i>Psme3</i> siRNA or BMDMs from CSS11(GM-CSF, IL-1β, IL-6, and TNF-α).
<p>(<b>A</b>) Down-regulation of <i>Dusp3</i> and <i>Psme3</i> by siRNA led to increased cytokine RNA expression upon <i>S. aureus</i> challenge in RAW264.7 macrophages. At three hours post-infection total RNA was extracted followed by reverse-transcription PCR and SYBR-Green qPCR. The expression of all genes were normalized to 18s rRNA. The expression level of GM-CSF, IL-1β, IL-6, and TNF-α was higher in <i>Dusp3</i> knockdown RAW cells, and the level of GM-CSF and IL-6 was higher in <i>Psme3</i> knockdown RAW cells. p-value smaller than 0.05 was considered significant. (<b>B</b>) BMDMs cytokine RNA production in CSS11 mice was significantly higher than in C57BL/6J upon <i>S. aureus</i> infection. 2×10<sup>6</sup> BMDMs were seeded to single wells in a 6-well plate the day before infection. At three hours post-infection, RNA was extracted using RNeasy followed by RT-PCR and qPCR. The expression levels of GM-CSF, IL-1β, IL-6, and TNF-α were significantly higher in BMDMs from CSS11 mice. The expression of all genes were normalized to 18s rRNA. p-value smaller than 0.05 was considered significant. (<b>C</b>) Down-regulation of <i>Dusp3</i> and <i>Psme3</i> by siRNA led to increased cytokine RNA expression upon <i>S. aureus</i> challenge in BMDMs of C57BL/6J. The expression level of IL-6 was higher in <i>Dusp3</i> siRNA transfected BMDMs, and the expression of TNF-α was higher in both <i>Dusp3</i> and <i>Psme3</i> siRNA transfected BMDMs.</p
