residual PFA. Treated bacterial suspensions were plated on chocolate II agar to ensure effective killing. Pulse-chase infections were done by infecting for 2 hours, removing the media, washing the cells with sterile PBS, and then incubating in media containing 50 mg/ml gentamicin (Invitrogen, Carlsbad, CA) for 30 minutes. Cells were then washed again and incubated in media containing 10 mg/ml gentamicin for the duration of the experiments. In Vivo Challenge with F.n. Wild-type mice were injected intraperitoneally with either 200 CFU of F.n. or PBS in accordance with institutional animal use protocols. The infectious dose was verified by CFU assays. Mice were euthanized at 24, 48 or 72 hours post-infection. The liver, lungs, and spleen were harvested, passed though a 70 mm cell strainer (BD, Bedford, MA), centrifuged briefly and then resuspended in TRIzolH reagent (Invitrogen, Carlsbad, CA). RNA extraction was then performed according to manufacturer instructions (Invitrogen). ELISA Cytokine Measurements Sandwich ELISAs were done for human TNFa using kits from R&D Systems (Minneapolis, MN) and for human IL-1b using kits from eBioscience (San Diego, CA) as done previously [17]. Western Blot Analysis Cells were lysed in TN1 buffer (50 mM Tris [pH 8.0], 10 nm EDTA, 10 M Na4P2O7, 10 nM NaF, 1% Triton-X 100, 125 nM NaCl, 10 nM Na3VO4, 10 mg/ml of both aprotinin and leupeptin. Proteins were electrophoretically separated on 10% acrylamide gels, tranferred to nitrocellulose membranes and then probed with rabbit polyclonal anti-mouse SHIP (generously provided by Dr. K. Mark Coggeshall) or anti-human SHIP (Upstate Cell Signaling Solution, Lake Placid, NY). Goat polyclonal antibody against actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit antibodies against pIKKa and NFkBp65 were purchased from Cell Signaling (Beverly, MA). Detections were performed using HRP-conjugated secondary antibodies followed by en- hanced chemiluminescence (GE, Buckinghamshire, UK) as previously described [17]. Real-Time PCR Cells were lysed in TRIzolH reagent (Invitrogen, Carlsbad, CA) and RNA isolation was completed according to the manufacturer’s instructions. 10 to 100 ng of total RNA were used for reverse transcription as measured by a ND-1000 spectrophotometer (Nanodrop, Wilmington, DE). cDNA was made using the TaqManH MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) and the specific reverse transcription primers for hsa-miR-155, mmu-miR-155, and the housekeeping genes RNU44 and RNU48 (human), sno412 and sno202 (mouse) (Applied Biosystems, Foster City, CA). The corresponding PCR primers were used with TaqManH Universal PCR Master Mix, No AmpEraseH UNG (Applied Biosystems, Branchburg, NJ) using an ABI PRISM 7900HT Fast PCR system. cDNA for BIC mRNA expression was primed for reverse transcription with 0.8 nM of random hexamer (Applied Biosystems, Foster City, CA) and analyzed by qRT-PCR using custom Taqman probes spanning References MiR155 Regulates Host Response exons 2–3 (Hs01374570) (Applied Biosystems, Foster City, CA). Control reactions containing no reverse transcriptase and no cDNA template were also performed. Samples were measured in triplicate for each experiment, and each experiment was performed at least 3 times. Relative expression was calculated as 2ˆ 2(CT Target2CT Housekeeping Gene) and significance was determined by a paired Student t test [26]. To simplify data presentation, relative expression values were converted to fold change over uninfected. Transfection 5 mg of pcDNA3.1 vector with or without exon 3 of human BIC was used for each transfection with 106106 PBM. Amaxa solution T was used for electroporation using program Y-01 as previously described [18]. Infections were performed 14 hours post-transfec- tion. Optimal miR-155 expression was found between 12 and 14 hours post-transfection by qRT-PCR analysis. Cell viability was monitored by trypan blue staining. At this time approximately 70% of the original PBM were recovered/viable and the numbers were comparable in vector versus miR-155 transfected cells. Construction of psiCHECK/INPP5D A fragment encompassing the INPP5D 39UTR was PCR- amplified using the forward primer (59-.AGC CCT CAG TGA GCT GCC ACT GAG TCG -.39) and reverse primer (59- .GAG TGA GAA AGG CAC AAT TTA ATT GG-.39). This was subcloned into the PCR2.1 vector following the manufactur- er’s protocol (Invitrogen). Plasmid DNA was isolated from transformed colonies and verified by dideoxy chain termination sequencing. The fragment was removed from the PCR2.1 plasmid by digestion with EcoRI and blunt-end ligated into the psiCHECK vector downstream of the f-luc reporter gene. The final construct was verified by dideoxy chain termination sequencing. Luciferase Reporter Assay Dual-luciferase reporter assays were done using the psiCHECK vector with or without the 39 UTR of SHIP, cotransfected with 50 ng of the pRL-CMV Renilla luciferase vector (Promega, Madison, WI). CHO cells were transfected with 0 to 50 nM synthetic miR precursor of miR-155 or scrambled miR control (Ambion) and luciferase reporter vectors using lipofectamine 2000 (Invitrogen, Carlsbad, CA). 48 hours post transfection cells were lysed with passive lysis buffer (Promega) and dual-luciferase activity was assayed using a luminometer as previously described [26]. Acknowledgments We thank Dr. S. Akira (Osaka University) for the MyD88-deficient mice and Dr. Amy Hise (Case Western University) for caspase-1-deficient mice. We thank Donna Cain and Yufen Wang for assistance with human monocyte isolation and BMM cultures. Author Contributions Conceived and designed the experiments: TJC CBM TSE AOA JPB ST. Performed the experiments: TJC DHR CDC TSE JPB. Analyzed the data: TJC DHR MGPH TSE AOA LSS JPB ST. Contributed reagents/ materials/analysis tools: TSE JSG TDK. Wrote the paper: TJC JPB ST. 1. Lofgren S, Tarnvik A, Bloom GD, Sjoberg W (1983) Phagocytosis and Killing of Francisella-Tularensis by Human Polymorphonuclear Leukocytes. Infection and Immunity 39: 715–720. 2. Lai XH, Golovliov I, Sjostedt A (2001) Francisella tularensis induces cytopathogenicity and apoptosis in murine macrophages via a mechanism that requires intracellular bacterial multiplication. Infection and Immunity 69: 4691–4694. 3. Clemens DL, Horwitz MA (2007) Uptake and intracellular fate of Francisella tularensis in human macrophages. Francisella Tularensis: Biology, Pathogenic- ity, Epidemiology, and Biodefense. Ann N Y Acad Sci 1105: 160–186. PLoS ONE | www.plosone.org 10 December 2009 | Volume 4 | Issue 12 | e8508