And secretion of interleukin 1 by way of Ipaf. Nature Immunology. 2006; 7:56975. [PubMed: 16648853] 27. Decker T, Lohmann-Matthes ML. A fast and basic process for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis issue (TNF) activity. J. Immunol. Strategies. 1988; 115:619. [PubMed: 3192948] 28. Warren SE, et al. Cutting Edge: Cytosolic Bacterial DNA Activates the Inflammasome by way of Aim2. The Journal of Immunology. 2010; 185:81821. [PubMed: 20562263] 29. Kitamura T, et al. Retrovirus-mediated gene transfer and expression cloning: effective tools in functional genomics. Exp Hematol. 2003; 31:1007014. [PubMed: 14585362]NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptCCR5 Gene ID Science. Author manuscript; available in PMC 2014 September 13.Hagar et al.SGLT1 list PageNIH-PA Author Manuscript NIH-PA Author ManuscriptFig. 1. Cytoplasmic LPS triggers caspase-11 activation(A ) BMM had been LPS primed overnight prior to transfection. (A ) BMMs had been transfected with the indicated bacterial lysates packaged in Lipofectamine 2000. Cytotoxicity was determined by lactate dehydrogenase release 4 hours later. Exactly where indicated, lysates have been treated with RNase, DNase, proteinase K, and lysozyme (RDLP) (B) or ammonium hydroxide (C). (D ) BMMs had been transfected with ultrapure LPS from S. minnesota RE595 packaged with DOTAP, a liposomal transfection reagent. Cytotoxicity (D) and IL-1 secretion by ELISA (E) were determined 4 h post transfection. (F ) BMMs were stimulated as in (D) and caspase-1 and -11 processing by western blot were examined 2 h post transfection. (H) Immortalized Casp1-/-Casp11-/- BMMs (iBMMs) complemented by retroviral transduction of Casp1 or Casp11 have been transfected with LPS from S. minnesota RE595. Cytotoxicity was determined immediately after four h. (I) Macrophages had been primed overnight with LPS (50ng/mL), poly(I:C) (1 /mL), IFN- (8ng/mL), or left untreated. Cells have been then transfected with LPS from S. minnesota RE595 and cytotoxicity was determined two h later. Data are representative of at least 3 experiments. Error bars indicate typical deviation of technical replicates.NIH-PA Author ManuscriptScience. Author manuscript; obtainable in PMC 2014 September 13.Hagar et al.PageNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptFig. 2. Listeria and CTB mediate caspase-11 activation by LPS(A ) The indicated macrophages had been primed with poly(I:C) or LPS and after that infected by L. monocytogenes (MOI 5) in the presence or absence of LPS from S. minnesota RE595 (1 /mL). Cytotoxicity (A, C, D), IL-1 secretion (B), or caspase-1 and caspase-11 processing (E ) had been examined 4 h post-infection. (G) Poly(I:C) and Pam3CSK4 primed macrophages were incubated using the indicated combinations of CTB (20 /mL), LPS from E. coli O111:B4 (1 /mL), and PrgJ (ten /mL). Cytotoxicity was determined 16 h later. Data are representative of 3 (A, D, G) or two (B, C, E, F) experiments. Error bars indicate common deviation of technical replicates.Science. Author manuscript; accessible in PMC 2014 September 13.Hagar et al.PageNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptFig. three. Caspase-11 responds to distinct lipid A structures(A) Poly(I:C) primed BMMs have been transfected with LPS from S. minnesota RE595 or S. typhimurium lipid A. Cytotoxicity was determined after two h. (B) Cytotoxicity in LPS primed BMMs was determined 4 hours just after infection with F. novicida (MOI 200). (C) LPS primed BMMs have been tr.