Boston, MA), followed by ECL detection (GE Healthcare, Buckinghamshire, UK). Real-Time RT-PCR Splenocytes from Brucella infected and non-infected 129Sv/Ev and IFN-abR KO mice or BMDM cells from 129Sv/Ev, IFN- abR KO, C57BL/6, TLR2 KO, TLR4 KO, TLR9 KO, MyD88 KO and TRIF KO mice were snap-frozen and stored in 280uC freezer until RNA isolation. Cells were homogenized in 350 mL of RA1 buffer and 3.5 mL of b-mercaptoethanol (illustraTM RNAspin Mini RNA Isolation Kit – GE Healthcare, Buckinghamshire, UK) and total RNA was isolated according to the manufacter’s instruction. Reverse transcription of 1 mg from total RNA was performed using illustraTM Ready-To-Go RT-PCR Beads (GE Healthcare, Buckinghamshire, UK). Real-Time RT-PCR was conducted in a final volume of 10 mL containing the following: SYBRH Green PCR Master Mix (Applied Biosystems, Foster City, CA), oligo-dT cDNA as the PCR template and 20 mM of primers. The PCR reaction was performed with ABI 7900 Real-Time PCR System (Applied Biosystems, Foster City, CA), using the following cycling parameters: 60uC for 10 min, 95uC for 10 min, 40 cycles of 95uC for 15 sec and 60uC for 1 min, and a dissociation stage of 95uC for 15 sec, 60uC for 1 min, 95uC for 15 sec, 60uC for 15 sec. Primers were used to amplify a specific 100–120-bp fragment corresponding to specific gene targets as follows: IFN-b F: 59- AGCTCCAAGAAAGGACGAACAT-39, IFN-b R: 59-GCCCT- GTAGGTGAGGTTGATCT-39, TRAIL F: 59-ACCTCAGCTT- CAGTCAGCACTTC-39, TRAIL R: 59-TGTAAGTCACAGC- CACAGACACAG-39, b-Actin F: 59-AGGTGTGCACCTTT- TATTGGTCTCAA-39, b-Actin R: 59-TGTATGAAGGTTTGG- TCTCCCT-39; IRF3 F: 59-GGACTTGCACATCTCCAACA-39, IRF3 R: 59-TTGCTCCACGTAGGGACAAT-39, STING F: 59- CCTAGCCTCGCACGAACTTG-39, STING R: 59-CGCA- CAGCCTTCCAGTAGC-39, IP10 F: 59-CCTGCCCACGT- GTTGAGAT-39, IP10 R: 59-TGATGGTCTTAGATTCCG- GATTC-39, IRF7 F: 59-CGCGGCACTAACGACAGGCGAG- 39, IRF7 R: 59-GCTGCCGTGCCCGGAATTCCAC-39. All data is presented as relative expression units after normalization to the b-actin gene. PCR measurements were conducted in triplicate. TdT-mediated dUTP Nick-End Labeling (TUNEL) and Annexin V assays Splenic tissue from infected and non-infected IFN-abR KO or 129 Sv/Ev mice were removed and fixed in 4% paraformaldehyde. Four mm sections were cut and mounted onto poly-l-lysine–coated slides. The samples were permeabilized using 0.1% Triton and 0.1% sodium citrate, and then double stranded DNA breaks were labeled using an in situ FragELTM DNA Fragmentation Detection Kit, Colorimetric - TdT Enzyme (Calbiochem, Darmstadt, Germany) according to the manufacturer’s protocol. KS300 software contained in Carl Zeiss image analyzer was used for image acquisition of 50 images randomly chosen from each lamina at a microscope with 406 objective lens and scanned through micro- camera JVC TK-1270/RGB. Then, the percentage of TUNEL positive cells was determined. For annexin V+ staining, splenocytes from Brucella infected and non-infected IFN-abR KO or 129 Sv/Ev mice were obtained 2 weeks after infection and they were washed twice with cold PBS and then ressuspended in 16 Binding Buffer (BD-Pharmingen, San Diego, CA). Cells were stained with PE- annexin V+/7-AAD2 as recommended by the manufacturer’s protocol (BD-Pharmingen, San Diego, CA). A minimum of 10,000 splenocyte-gated events were acquired using a FACScan apparatus (BD-Pharmingen, San Diego, CA). FACS results were analyzed using the FlowJo Software (Tree Star, Ashland, OR). IFN-b Induction by Brucella Requires MyD88/STING siRNA and RNA Polymerase III inhibition Primer sequences for silencing mouse IRF3 (accession number BC.050882.1) were predicted with siRNA Target Finder and siRNA Converter (AmbionH - Applied Biosystems, Foster City, CA) and further prepared with the Ambion SilencerTM siRNA kit. The primer sequences used were: AS-mIRF3-1075: 59-AAGG- TTGTTCCTACATGTCTTCCTGTC-39, SENSE-mIRF3-1075 59-AAAAGACATGTAGGAACAACCCCTGTCTC-39. For siRNA transfections, RAW 264.7 cells were cultured in 12-well plates, and transfections were performed using FuGENE 6 (Roche Applied Science, Indianapolis, IN) in mixture with 2 mg of siRNA per well. After 72 hours, the cells were stimulated with 103 bacteria per cell or with 10 mg of poly:IC (InvivoGen, San Diego, CA). Accell SMARTpool siRNA duplexes targeting mouse STING (catalog nu E-055528), and Accell siRNA delivery media were purchased from Dharmacon (Thermo Fischer Scientific, Lafay- ette, CO). For STING siRNA-mediated knockdown, RAW 264.7 cells were first pretreated with the indicated siRNA or control duplexes for 72 hours in Accell siRNA media. Then the culture medium was replaced and the cells stimulated with 103 bacteria/ cell, DNA (1 mg/well) or poly I:C (10 mg/mL; InvivoGen, San Diego, CA). Messenger RNA silencing was confirmed by quantitative SyBR-Green RT-PCR. Briefly, total RNA was extracted with TRIzol (GIBCO BRL) and further reverse- transcribed into cDNA with Ready-To-Go RT-PCR Beads (GE Healthcare, Buckinghamshire, UK) and real-time PCR was performed as described. For RNA polymerase III studies, Raw 264.7 cells were pretreated with the Pol-III inhibitor, ML-60218 (Calbiochem, Darmstadt, Germany), at 0.1 mM or 1.0 mM for 10 hours and then stimulated with 103 bacteria/cell, 1 mg of purified Brucella DNA, or 10 mg/mL of poly I:C (InvivoGen, San Diego, CA) for 3 hours. The supernatants were collected and the total RNA was extracted from the cells to access the expression of IFN-b by real-time RT-PCR as described above. Statistical analysis The differences in IFN-b, IFN-c, IL-17 and NO levels between wild type and IFN-abR KO mice were analyzed by Student’s t test using two-tail distribution. The differences in the relative expression of target genes were analyzed by Student’s t test, using one tail distribution and heteroscedastic variance. Supporting Information Figure S1 Lack of type I IFN signaling is not related to IL-17A production in Brucella-infected splenocytes. Spleen cells from B. abortus S2308 infected 129Sv/Ev or IFN- abR KO mice after two-weeks of infection were cultured with 102 bacteria/cell, ConA (5 mg/ml) or medium alone for 72 hrs and IL- 17A was detected by ELISA. Error bars represent the mean 6SD. Similar results were obtained in two-independent experiments. Statistically significant difference from medium alone compared to bacteria or ConA is denoted by an asterisk (p,0.05). (TIF) Figure S2 Brucella DNA but not its LPS or lipidated- Omp19 induces IFN-b expression in macrophages. BMDM from C57BL/6, TLR2 or TLR4 KO mice were infected with B. abortus strain S2308 (103 bact/cell), stimulated with Brucella DNA (1 mg/56105 cells), Brucella DNA treated with DNAse (0.2 U/mg DNA), poly I:C (10 mg/ml), Brucella LPS (10 mg/ml), Brucella L-Omp19 (25 mg/ml) or medium alone. Twenty four hours after stimulation of the cells, total RNA was harvested and mRNA levels of IFN-b was determined by real-time RT-PCR and normalized to b-actin. Error bars represent the mean 6SD of PLoS ONE | www.plosone.org 12 August 2011 | Volume 6 | Issue 8 | e23135