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 inside 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. Actually, direct labeling of cells in conditioned culture medium led to robust staining with the nucleoli (Fig. S2C). Of note, EU labeling was performed in starvation medium that facilitates the incorporation of EU. Indeed, EU isn’t quantitatively incorporated in host RNA when cultured in typical culture medium for this quick time span (Fig. S2D). Given that we cultivated cell lines in typical culture medium for the duration of infection, we moreover excluded the possibility that host cell RNA is stained by nonincorporated EU released from L. monocytogenes. From the data we conclude that through infection, significant amounts of bacRNA are transferred in to the cytosol of cells where it could be detected by cytosolic immune receptors. THP1 cells infected with L. monocytogenes lacking the secA2 secretion method, which was not too long ago identified to become accountable for secretion of bacterial RNA of L. monocytogenes, had been 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 inside a wide range of cell lines and cell sorts. Extra recent studies exhibited that in human cell lines this refers only towards the recognition of ATrich [23,55] sequences. Extended ATrich DNA sequences (poly(dAdT)) were discovered to be the template of RNA polymerase III (pol III) major to synthesis of triphosphorylated RNA, the ligand of RIGI [23,24]. Nonetheless, nonAT wealthy dsDNA molecules inside the size array of poly(dAdT) nonetheless induced a variety I IFN response in human monocytederived dendritic cells (MoDC) in which RIGI is silenced [23]. 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) type I IFN induction by long random DNA. This redundancy in DNA sensing mechanisms complicates the delineation of pattern recognition receptors involved in L. monocytogenes sensing. Current data suggested that the STING dependentPLOS 1 | www.plosone.orgRIGI Detects RNA of Listeria in NonImmune CellsFigure two.3-Bromo-1-naphthoic acid Chemical name RNA of L.39692-67-6 site monocytogenes has access towards the cytosol on the host cell during infection.PMID:23376608 THP1, A549 and HepG2 had been infected with FITCtagged and EUlabeled wt and hly L. monocytogenes for the indicated duration. Cells were 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. Ideal column, hly L. monocytogenes infection 4 hrs. A: THP1 cells. B: A549 cells. C: HepG2 cells. As determined by counting of single bacteria in cells (50 cells per slidePLOS One particular | www.plosone.orgRIGI Detects RNA of Listeria in NonImmune Cellswere counted) the average bacterial load was 9(wt) and 4(hly) bacteria per cell for THP1 cells, 6(wt) and 4(hly) bacteria per cell for A549 cells and six(wt) and five(hly) bacteria per cell for HepG2 cells, one repres.