Ipt NIH-PA Author Manuscript NIH-PA Author ManuscriptScience. Author manuscript; obtainable in PMC 2014 September 13.Hagar et al.PageNIH-PA Author Manuscript NIH-PA Author ManuscriptFig. 1. Cytoplasmic LPS triggers caspase-11 activation(A ) BMM were LPS primed overnight prior to transfection. (A ) BMMs have been transfected with all the indicated bacterial lysates packaged in Lipofectamine 2000. Cytotoxicity was determined by lactate dehydrogenase release 4 hours later. Where indicated, lysates have been treated with RNase, DNase, proteinase K, and lysozyme (RDLP) (B) or ammonium hydroxide (C). (D ) BMMs were 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 four h post transfection. (F ) BMMs were stimulated as in (D) and caspase-1 and -11 processing by western blot were examined two h post transfection. (H) Immortalized Casp1-/-Casp11-/- BMMs (iBMMs) complemented by retroviral transduction of Casp1 or Casp11 had been transfected with LPS from S. minnesota RE595. Cytotoxicity was determined soon after 4 h. (I) Macrophages were primed overnight with LPS (50ng/mL), poly(I:C) (1 /mL), IFN- (8ng/mL), or left untreated. Cells had been then transfected with LPS from S. minnesota RE595 and cytotoxicity was determined two h later. Data are representative of at least three experiments. Error bars indicate normal deviation of technical replicates.NIH-PA Author ManuscriptScience. Author manuscript; available in PMC 2014 September 13.Hagar et al.PageNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptFig. two. Listeria and CTB mediate caspase-11 activation by LPS(A ) The indicated macrophages have been primed with poly(I:C) or LPS and then infected by L. monocytogenes (MOI five) 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 ) have been examined 4 h post-infection. (G) Poly(I:C) and Pam3CSK4 primed macrophages have been incubated with the indicated combinations of CTB (20 /mL), LPS from E. coli O111:B4 (1 /mL), and PrgJ (10 /mL). Cytotoxicity was determined 16 h later. Data are representative of three (A, D, G) or two (B, C, E, F) experiments. Error bars indicate regular deviation of technical replicates.Science. Author manuscript; out there in PMC 2014 September 13.Hagar et al.PageNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptFig. 3. Caspase-11 responds to distinct lipid A structures(A) Poly(I:C) primed BMMs had been transfected with LPS from S. minnesota RE595 or S. typhimurium lipid A. Cytotoxicity was determined following two h.1053656-57-7 Order (B) Cytotoxicity in LPS primed BMMs was determined four hours just after infection with F.3-(4-Fluorophenoxy)azetidine manufacturer novicida (MOI 200).PMID:25023702 (C) LPS primed BMMs had been transfected with mock or DNase treated F. novicida lysates. Cytotoxicity was determined four hours later. (D) Macrophages have been infected as in (B) inside the presence or absence of LPS from S. minnesota RE595. (E) Structural comparison of lipid A from wild variety F. novicida or the lpxF mutant. Structural alterations are indicated. (F) Poly(I:C) primed macrophages were transfected with lipid A from F. novicida grown at 18 or 37 , or theScience. Author manuscript; obtainable in PMC 2014 September 13.Hagar et al.Pageindicated F. novicida mutants grown at 37 . Cytotoxicity was determined right after 2 h. (G) Structural comparison of lipid A from Y. pestis grown at 25 or 37 . (H) Poly(I:C) primed BMMs have been infe.