Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IRF8 regulates Gram-negative bacteria–mediated NLRP3 inflammasome activation and cell death

doi: 10.4049/jimmunol.1901508

Figure Lengend Snippet: (A) Immunoblot analysis of pro-caspase-1 (CASP1) (p45) and cleaved CASP1 (p20) in wild-type (WT) or Irf8–/– bone marrow-derived macrophages (BMDMs) infected with E. coli (multiplicity of infection [MOI], 20) for the indicated time. (B) Assessment of IL-18 and IL-1β release by ELISA in BMDMs 16 h after E. coli infection. (C) Real time cell death analysis by Incucyte and SYTOX green staining in BMDMs infected with E. coli for the indicated time. (D) Representative images of BMDMs under light microscopy after E. coli infection for 16 h. The arrows indicate pyroptotic cells. (E) Immunoblot analysis of pro-CASP1 (p45) and cleaved CASP1 (p20) in WT or Irf8–/– BMDMs infected with C. rodentium (MOI, 20) for the indicated time. (F) Assessment of IL-18 and IL-1β release by ELISA in BMDMs 16 h after C. rodentium infection. (G) Immunoblot analysis of pro-CASP1 (p45) and cleaved CASP1 (p20) in WT or Irf8–/– BMDMs infected with P. aeruginosa ΔpopB (MOI, 50) for the indicated time. (H) Assessment of IL-18 and IL-1β release by ELISA in BMDMs 16 h after P. aeruginosa ΔpopB infection. *P < 0.05, **P < 0.01, and ***P < 0.001 (one-way ANOVA with Dunnett’s multiple comparisons test). Data are representative of three (A, D, E, and G) experiments or are from three (B, C, F, and H) independent experiments (mean ± SEM).

Article Snippet: Citrobacter rodentium (51549, American Type Culture Collection), Escherichia coli (11775, American Type Culture Collection), and the isogenic mutant of Pseudomonas aeruginosa strain, Δ popB (provided by Dr. Brent Berwin, Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon), were inoculated into Luria-Betrani (LB) media (3002–031, MP Biomedicals) and incubated overnight under aerobic conditions at 37°C.

Techniques: Western Blot, Derivative Assay, Infection, Enzyme-linked Immunosorbent Assay, Staining, Light Microscopy

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IRF8 regulates Gram-negative bacteria–mediated NLRP3 inflammasome activation and cell death

doi: 10.4049/jimmunol.1901508

Figure Lengend Snippet: (A) Immunoblot analysis of NLRP3, apoptosis associated speck-like protein containing a caspase activation and recruitment domain (ASC), pro-IL-1β, caspase-11 (CASP11), and gasdermin D (GSDMD) in wild-type (WT) or Irf8–/– bone marrow-derived macrophages (BMDMs) infected with E. coli (multiplicity of infection [MOI], 10) for the indicated time. (B) Immunoblot analysis of NLRP3, ASC, pro-IL-1β, CASP11, and GSDMD in WT or Irf8–/– BMDMs infected with C. rodentium (MOI, 10) for the indicated time. (C) Immunoblot analysis of NLRP3, ASC, pro-IL-1β, CASP11, and GSDMD in WT or Irf8–/– BMDMs infected with P. aeruginosa ΔpopB (MOI, 10) for the indicated time. (D) Immunoblot analysis of phospho (p)-IκBα and IκBα in WT or Irf8–/– BMDMs infected with E. coli (MOI, 10) for the indicated time. GAPDH was used as an internal control. Data are representative of three independent experiments.

Article Snippet: Citrobacter rodentium (51549, American Type Culture Collection), Escherichia coli (11775, American Type Culture Collection), and the isogenic mutant of Pseudomonas aeruginosa strain, Δ popB (provided by Dr. Brent Berwin, Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon), were inoculated into Luria-Betrani (LB) media (3002–031, MP Biomedicals) and incubated overnight under aerobic conditions at 37°C.

Techniques: Western Blot, Activation Assay, Derivative Assay, Infection, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IRF8 regulates Gram-negative bacteria–mediated NLRP3 inflammasome activation and cell death

doi: 10.4049/jimmunol.1901508

Figure Lengend Snippet: (A–C) The expression of Ifnb in wild-type (WT) or Irf8–/– bone marrow-derived macrophages (BMDMs) at 2, 4, and 6 h after infection with E. coli (multiplicity of infection [MOI], 10), C. rodentium (MOI, 10), or P. aeruginosa ΔpopB (MOI, 10). (D) Assessment of IFN-β release by ELISA in WT or Irf8–/– BMDMs 12 h after E. coli (MOI, 10), C. rodentium (MOI, 10), or P. aeruginosa ΔpopB (MOI, 10) infection. (E) Assessment of IFN-β release by ELISA in WT or Irf8–/– BMDMs 9 h after stimulation with LPS (100 ng/mL) or poly(I:C) (10 μg/mL) in the media. (F,G) Immunoblot analysis of pro- and cleaved caspase-1 (CASP1) and gasdermin D (GSDMD), and the assessment of IL-18 release in BMDMs pre-treated with IFN-β (400 U/mL) for 4 h followed by infection with E. coli (MOI, 20) for an additional 16 h. NS, not significant; ***P < 0.001 and ****P < 0.0001 (D and E, one-way ANOVA with Sidak’s multiple comparisons test and G, two-way ANOVA with Tukey’s multiple comparisons test). Data are representative of three (F) experiments or are from three (A–E and G) independent experiments (mean ± SEM).

Article Snippet: Citrobacter rodentium (51549, American Type Culture Collection), Escherichia coli (11775, American Type Culture Collection), and the isogenic mutant of Pseudomonas aeruginosa strain, Δ popB (provided by Dr. Brent Berwin, Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon), were inoculated into Luria-Betrani (LB) media (3002–031, MP Biomedicals) and incubated overnight under aerobic conditions at 37°C.

Techniques: Expressing, Derivative Assay, Infection, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IRF8 regulates Gram-negative bacteria–mediated NLRP3 inflammasome activation and cell death

doi: 10.4049/jimmunol.1901508

Figure Lengend Snippet: (A) Immunoblot analysis of pro- and cleaved caspase-11 (CASP11), caspase-8 (CASP8), caspase-3 (CASP3), and caspase-7 (CASP7) in WT or Irf8–/– bone marrow-derived macrophages (BMDMs) infected with E. coli (multiplicity of infection [MOI], 20) for the indicated time. (B) Immunoblot analysis of pro- and cleaved CASP11, CASP8, CASP3, and CASP7 in WT or Irf8–/– BMDMs infected with C. rodentium (MOI, 20) for the indicated time. (C) Immunoblot analysis of pro- and cleaved CASP11, CASP8, and CASP7 in WT or Irf8–/– BMDMs infected with P. aeruginosa ΔpopB (MOI, 50) for the indicated time. Data are representative of three independent experiments.

Article Snippet: Citrobacter rodentium (51549, American Type Culture Collection), Escherichia coli (11775, American Type Culture Collection), and the isogenic mutant of Pseudomonas aeruginosa strain, Δ popB (provided by Dr. Brent Berwin, Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon), were inoculated into Luria-Betrani (LB) media (3002–031, MP Biomedicals) and incubated overnight under aerobic conditions at 37°C.

Techniques: Western Blot, Derivative Assay, Infection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IRF8 regulates Gram-negative bacteria–mediated NLRP3 inflammasome activation and cell death

doi: 10.4049/jimmunol.1901508

Figure Lengend Snippet: (A,B) Immunoblot analysis of phospho (p)-IRF3, IRF3, p-TBK1, TBK1, p-IKKε, and IKKε in wild-type (WT) or Irf8–/– bone marrow-derived macrophages (BMDMs) infected with E. coli (multiplicity of infection [MOI], 10) and C. rodentium (MOI, 10) for the indicated time. GAPDH was used as an internal control. Data are representative of three experiments.

Article Snippet: Citrobacter rodentium (51549, American Type Culture Collection), Escherichia coli (11775, American Type Culture Collection), and the isogenic mutant of Pseudomonas aeruginosa strain, Δ popB (provided by Dr. Brent Berwin, Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon), were inoculated into Luria-Betrani (LB) media (3002–031, MP Biomedicals) and incubated overnight under aerobic conditions at 37°C.

Techniques: Western Blot, Derivative Assay, Infection, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IRF8 regulates Gram-negative bacteria–mediated NLRP3 inflammasome activation and cell death

doi: 10.4049/jimmunol.1901508

Figure Lengend Snippet: (A) Immunoblot analysis of interferon regulatory factor 1 (IRF1), guanylate binding protein 2 (GBP2), and GBP5 in wild-type (WT) or Irf8–/– bone marrow-derived macrophages (BMDMs) after E. coli (multiplicity of infection [MOI], 10) or C. rodentium (MOI, 10) infection. (B) Immunoblot analysis of IRGB10 and IRF8 in WT or Irf8–/– BMDMs after E. coli (MOI, 10) infection. (C) Immunoblot analysis of phospho (p)-STAT1 and STAT1 in WT or Irf8–/– BMDMs after E. coli (MOI, 10) or C. rodentium (MOI, 10) infection. (D) Immunoblot analysis of GBP2 and GBP5 WT, Irf8–/–, or Stat1–/– BMDMs after E. coli (MOI, 10) or C. rodentium (MOI, 10) infection. (E) Immunoblot analysis of pro-caspase-1 (CASP1) (p45) and cleaved CASP1 (p20) in WT, Irf8–/–, Stat1–/–, or Casp11–/– BMDMs infected with C. rodentium (MOI, 20) for 16 h. Data are representative of three independent experiments.

Article Snippet: Citrobacter rodentium (51549, American Type Culture Collection), Escherichia coli (11775, American Type Culture Collection), and the isogenic mutant of Pseudomonas aeruginosa strain, Δ popB (provided by Dr. Brent Berwin, Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Lebanon), were inoculated into Luria-Betrani (LB) media (3002–031, MP Biomedicals) and incubated overnight under aerobic conditions at 37°C.

Techniques: Western Blot, Binding Assay, Derivative Assay, Infection

Journal: The ISME Journal

Article Title: The soil microbial food web revisited: Predatory myxobacteria as keystone taxa?

doi: 10.1038/s41396-021-00958-2

Figure Lengend Snippet: Myxobacteria and potential prey organisms used in predation assays.

Article Snippet: The myxobacteria Haliangium ochraceum , Stigmatella aurantiaca , Kofleria flava , Corallococcus coralloides, and Chondromyces robustus were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Table ).

Techniques: Bacteria