Immunology. 2010; 185:81821. [PubMed: 20562263] 29. Kitamura T, et al. Retrovirus-mediated gene transfer and expression

Immunology. 2010; 185:81821. [PubMed: 20562263] 29. Kitamura T, et al. Retrovirus-mediated gene transfer and expression cloning: strong tools in functional genomics. Exp Hematol. 2003; 31:1007014. [PubMed: 14585362]NIH-PA Author Manuscript 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 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 were treated with RNase, DNase, proteinase K, and lysozyme (RDLP) (B) or ammonium hydroxide (C). (D ) BMMs had been transfected with ultrapure LPS from S.Lazertinib minnesota RE595 packaged with DOTAP, a liposomal transfection reagent.LB-100 Cytotoxicity (D) and IL-1 secretion by ELISA (E) have been determined 4 h post transfection. (F ) BMMs were stimulated as in (D) and caspase-1 and -11 processing by western blot had been examined two h post transfection. (H) Immortalized Casp1-/-Casp11-/- BMMs (iBMMs) complemented by retroviral transduction of Casp1 or Casp11 were transfected with LPS from S. minnesota RE595. Cytotoxicity was determined soon 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 had been then transfected with LPS from S. minnesota RE595 and cytotoxicity was determined two h later. Data are representative of no less than three experiments. Error bars indicate normal deviation of technical replicates.NIH-PA Author ManuscriptScience. Author manuscript; readily available in PMC 2014 September 13.Hagar et al.PageNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptFig.PMID:32695810 2. Listeria and CTB mediate caspase-11 activation by LPS(A ) The indicated macrophages were primed with poly(I:C) or LPS after which infected by L. monocytogenes (MOI 5) inside 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 ) were examined four h post-infection. (G) Poly(I:C) and Pam3CSK4 primed macrophages had been 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. Information are representative of 3 (A, D, G) or 2 (B, C, E, F) experiments. Error bars indicate standard deviation of technical replicates.Science. Author manuscript; obtainable 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 have been transfected with LPS from S. minnesota RE595 or S. typhimurium lipid A. Cytotoxicity was determined after 2 h. (B) Cytotoxicity in LPS primed BMMs was determined four hours after infection with F. novicida (MOI 200). (C) LPS primed BMMs were transfected with mock or DNase treated F. novicida lysates. Cytotoxicity was determined 4 hours later. (D) Macrophages have been infected as in (B) within the presence or absence of LPS from S. minnesota RE595. (E) Structural comparison of lipid A from wild sort 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; avai.