Bacterial viability contributes to miR-155 induction through the TLR pathway.

<p>(A) PBM were infected with live, heat-killed (HK), or paraformaldehyde-fixed (PFA) <i>F.n.</i> at an MOI of 50 for 24 hours, then miR-155 measured by qRT-PCR. (B) PBM were infected with live <i>F.n.</i> or heat-killed <i>F.n.</i> at an MOI of 50, then with or without gentamicin after 2 hours, then miR-155 was assayed by qRT-PCR. (C) Wild-type (WT) or MyD88^−/− bone marrow-derived macrophages were infected with <i>F.n.</i> at an MOI of 50 for 24 hours, then relative miR-155 expression measured by qRT-PCR and then converted to fold change over uninfected. (D) ELISA for TNFα secretion in supernatants from the samples in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008508#pone-0008508-g003" target="_blank">Figure 3C</a>. (E) Wild-type (WT) and TLR2 (TLR2 Mut) signaling mutant bone marrow-derived macrophages were infected with <i>F.n.</i> at an MOI of 50 for eight hours and miR-155 was assayed by qRT-PCR. (F) WT and TLR2 Mut macrophages were infected at an MOI of 50 of <i>F.n.</i> for 5, 20, 40, and 60 minutes. Cell lysates were analyzed by Western blotting for pIKKα, pNFκBp65, and Actin. (G) PBM were pretreated for 30 minutes with inhibitors of PI3K (LY), ERK (UO), JNK (SP), p38 (SB), or with DMSO vehicle control, infected with <i>F.n.</i> at an MOI of 50 for 6 hours, then miR-155 was assayed by qRT-PCR. (H) PBM were pretreated with the IKK inhibitor BAY7085 (BAY) or DMSO vehicle control for 90 minutes, infected with <i>F.n.</i> at an MOI of 50 for 6 hours, then miR-155 was assayed by qRT-PCR. (I) ELISA for TNFα in supernatants from the samples in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008508#pone-0008508-g003" target="_blank">Figure 3F</a>. All experiments were performed in triplicate, and each experiment performed three times. Graphs represent mean and standard deviation. Data were analyzed by a paired Student <i>t</i> test. An asterisk (*) indicates a <i>p</i>-value<0.05.