Was extracted and labeled with Alexa594azide in vitro. The information confirm that EU was equally incorporated in wt and hly L. monocytogenes RNA (Fig. S2B). The absence of labeled RNA in the host cell nucleoli, the web site of ribosomal RNA production, excluded transfer of EU nucleotides from L. monocytogenes with subsequent incorporation into host RNA. The truth is, direct labeling of cells in conditioned culture medium led to robust staining on the nucleoli (Fig. S2C). Of note, EU labeling was performed in starvation medium that facilitates the incorporation of EU. Certainly, EU is just not quantitatively incorporated in host RNA when cultured in normal culture medium for this brief time span (Fig. S2D). Due to the fact we cultivated cell lines in regular culture medium through infection, we on top of that excluded the possibility that host cell RNA is stained by nonincorporated EU released from L. monocytogenes. From the data we conclude that during infection, considerable amounts of bacRNA are transferred into the cytosol of cells where it might be detected by cytosolic immune receptors. THP1 cells infected with L. monocytogenes lacking the secA2 secretion program, which was recently identified to be accountable for secretion of bacterial RNA of L. monocytogenes, were not labeled for EUcontaining RNA (Fig. S3), suggesting that translocation of bacterial RNA is rather mediated by active secretion than by lysis of bacteria [53]. Sort I IFN induction by L. monocytogenes in epithelial cells and hepatocytes is triggered by recognition of bacterial RNA. Function by Ishii et al. [54] suggested that cytosolic recognition of DNA represents an ubiquitous pathway expressed within a wide range of cell lines and cell sorts. Far more recent studies exhibited that in human cell lines this refers only for the recognition of ATrich [23,55] sequences. Long ATrich DNA sequences (poly(dAdT)) were found to be the template of RNA polymerase III (pol III) leading to synthesis of triphosphorylated RNA, the ligand of RIGI [23,24]. Nonetheless, nonAT rich dsDNA molecules inside the size array of poly(dAdT) still induced a variety I IFN response in human monocytederived dendritic cells (MoDC) in which RIGI is silenced [23].Ir[FCF3(CF3)ppy]2(dtbbpy)PF6 web This points to two distinct DNA recognition pathways in immune cells: pol III/RIGI dependent recognition of ATrich DNA and STING dependent (polIII/RIGI independent) kind I IFN induction by extended random DNA.5,6-Dichloropyridazin-3(2H)-one supplier This redundancy in DNA sensing mechanisms complicates the delineation of pattern recognition receptors involved in L. monocytogenes sensing. Current data recommended that the STING dependentPLOS One particular | www.plosone.orgRIGI Detects RNA of Listeria in NonImmune CellsFigure 2. RNA of L. monocytogenes has access towards the cytosol from the host cell in the course of infection.PMID:23880095 THP1, A549 and HepG2 were infected with FITCtagged and EUlabeled wt and hly L. monocytogenes for the indicated duration. Cells had been then fixed, stained with Alexa594azide and counterstained with DAPI. Left column, wt L. monocytogenes infection 1 hr. Middle column, wt L. monocytogenes infection 4 hrs. Suitable column, hly L. monocytogenes infection four hrs. A: THP1 cells. B: A549 cells. C: HepG2 cells. As determined by counting of single bacteria in cells (50 cells per slidePLOS 1 | www.plosone.orgRIGI Detects RNA of Listeria in NonImmune Cellswere counted) the typical bacterial load was 9(wt) and four(hly) bacteria per cell for THP1 cells, 6(wt) and four(hly) bacteria per cell for A549 cells and six(wt) and five(hly) bacteria per cell for HepG2 cells, one particular repres.