Journal: PLoS Pathogens

Article Title: Salmonella Typhimurium disrupts Sirt1/AMPK checkpoint control of mTOR to impair autophagy

doi: 10.1371/journal.ppat.1006227

Figure Lengend Snippet: (A) ATP levels in BMDMs upon S . Typhimurium was analyzed by mass spectrometry and the mass peak intensity is depicted in the graph as mean ± SEM, ***p≤0.001 (n = 6). (B) Intracellular NAD + and NADH levels form uninfected and S . Typhimurium-infected BMDMs were measured using NAD+/NADH assay kit. Bar graphs are expressed as mean ± SEM, ***p≤0.001 (n = 3). (C) Immunoblot analysis of AMPK, ACC and LKB1 expression upon S . Typhimurium infection in BMDMs cells. (D) Mean densitometric analysis of immunoblots is shown. Data are representative of 3 independent experiments. Bar graphs are expressed as mean ± SEM, ***p≤0.001 and **p≤0.01. (E) Confocal image showing AMPK-LKB1 (n = 4). ( F ) Pearson’s correlation coefficient of AMPK with LKB1 analyzed from 50 regions of interest (ROI). (G) AMPK-LysoTracker Red co-localization (n = 4). (H) Pearson’s correlation coefficient of AMPK with LysoTracker Red analyzed from 50 ROIs. (I) LKB1-LysoTracker Red co-localization in BMDMs upon S . Typhimurium infection n = 3. ( J ) Pearson’s correlation coefficient of LKB1-LAMP1 co-localization calculated by measuring minimum of 50 ROI using olympus fluoview fv1000 software. Scale bar = 10μm for microscopy images. (K) Total AMPK and LKB1 expression upon S . Typhimurium infection in BMDMs treated with concanamycinA (concA) or MG132. Western blots are representative of three experiments. (L) Mean densitometric data of protein expression were analyzed using NIH Image J software. Bar graphs are expressed as mean ± SEM, ns non-significant, ***p≤0.001 and **p≤0.01.

Article Snippet: Bone marrow derived macrophages (BMDMs) were prepared as described [ ] from C57BL/6J mice maintained and bred in the animal facility of Center for Molecular Medicine, University of Cologne.

Techniques: Mass Spectrometry, Infection, Nad NADH Assay, Western Blot, Expressing, Software, Microscopy

Journal: PLoS Pathogens

Article Title: Salmonella Typhimurium disrupts Sirt1/AMPK checkpoint control of mTOR to impair autophagy

doi: 10.1371/journal.ppat.1006227

Figure Lengend Snippet: (A) Confocal image of Sirt1 and LKB1 in BMDMs upon S . Typhimurium infection (n = 3). (B) Pearson’s correlation coefficient of Sirt1 with LKB1 calculated by measuring 42 ROIs. (C) Sirt1 was immunoprecipitated (IP) from uninfected and S . Typhimurium-infected BMDMs and the precipitated samples were immunoblotted (IB) for LKB1, AMPK and Sirt1 (n = 2). ( D ) Immunoblot of Sirt1, acetylated NFκB and GAPDH in BMDMs upon S . Typhimurium infection. Data shown are representative of 6 independent experiments. ( E ) Densitometric analysis of immunoblots. Bar graphs are expressed as mean ± SEM, ***p≤0.001 and **p≤0.01. ( F ) Immunofluorescence image of BMDMs stained for Sirt1 and S . Typhimurium (n = 4). (G) Quantitation of Sirt1-ST co-localization with SCVs. 100 SCVs were counted and expressed as percentage co-localization. Bar graphs are expressed as mean ± SEM, ***p≤0.001. ( H ) Sirt1-Lysotracker red co-localization in BMDMs upon S . Typhimurium infection (n = 4). (I) Pearson’s correlation coefficient of Sirt1 with Lysotracker red calculated by measuring minimum of 50 ROI. ( J ) Sirt1 expression upon S . Typhimurium infection in BMDMs treated with bafilomycinA (BafA), E64D, pepstatin A and calpeptin. (K) Sirt1 expression levels are quantified by densitometric analysis. Data shown are representative of 3 independent experiments. Bar graphs are expressed as mean ± SEM, ***p≤0.001 and **p≤0.01. (L) Sirt1 expression in nuclear (N) and cytoplasmic (C) fractions of BMDMs infected with S . Typhimurium. LaminB and GAPDH were used as housekeeping controls for nuclear and cytoplasmic fractions respectively (n = 2). Scale bar = 10μm for microscopical images.

Article Snippet: Bone marrow derived macrophages (BMDMs) were prepared as described [ ] from C57BL/6J mice maintained and bred in the animal facility of Center for Molecular Medicine, University of Cologne.

Techniques: Infection, Immunoprecipitation, Western Blot, Immunofluorescence, Staining, Quantitation Assay, Expressing

Journal: PLoS Pathogens

Article Title: Salmonella Typhimurium disrupts Sirt1/AMPK checkpoint control of mTOR to impair autophagy

doi: 10.1371/journal.ppat.1006227

Figure Lengend Snippet: (A) Immunoblot analysis of AKT activation upon S . Typhimurium infection in BMDMs. (B) The phosphorylated and total AKT amounts are quantified by densitometric analysis. Data shown are representative of at least 3 independent experiments. Bar graphs are expressed as mean ± SEM, ***p≤0.001, **p≤0.01 and *p≤0.05. (C) Protein expression of AKT, Sirt1, GAPDH and ACC from BMDMs pretreated with or without AKT inhibitor VIII prior to infection with S . Typhimurium. Western bots are representative of 3 independent experiments. (D) The phosphorylated AKT, ACC and Sirt1 amounts are quantified by densitometric analysis. Bar graphs are expressed as mean ± SEM, ***p≤0.001, **p≤0.01 and *p≤0.05. ( E ) Confocal immunofluorescence image showing Sirt1-LysoTracker Red co-localization in BMDMs pretreated with AKT inhibitor VIII followed by S . Typhimurium infection. BMDMs untreated with AKT inhibitor VIII but infected with S . Typhimurium for 4h is shown for comparison (n = 3). (F) Pearson’s correlation coefficient of Sirt1 with LysoTracker Red calculated by measuring 35 ROIs. (G) Sirt1- S . Typhimurium co-localization in BMDMs pretreated with AKT inhibitor VIII followed by S . Typhimurium infection (n = 3). Scale bar = 10μm for microscopical images. (H) Quantitation of LysoTracker Red co-localization with SCVs. 100 SCVs were counted and expressed as percentage co-localization. Bar graphs are expressed as mean ± SEM, ***p≤0.001.

Article Snippet: Bone marrow derived macrophages (BMDMs) were prepared as described [ ] from C57BL/6J mice maintained and bred in the animal facility of Center for Molecular Medicine, University of Cologne.

Techniques: Western Blot, Activation Assay, Infection, Expressing, Immunofluorescence, Comparison, Quantitation Assay

Journal: PLoS Pathogens

Article Title: Salmonella Typhimurium disrupts Sirt1/AMPK checkpoint control of mTOR to impair autophagy

doi: 10.1371/journal.ppat.1006227

Figure Lengend Snippet: ( A ) Immunoblot analysis of S . Typhimurium-infected BMDMs for mTOR and its downstream targets p70S6K and NDRG1. (B) Densitomertic analysis of phosphorylation amounts of mTOR, p70s6K and NDRG1. Data shown are representative of at least 3 independent experiments. Bar graphs are expressed as mean ± SEM, ***p≤0.001, **p≤0.01 and *p≤0.05. (C) Confocal image of Sirt1- S . Typhimurium. (D) Quantitation of LysoTracker Red co-localization with SCVs. 100 SCVs were counted and expressed as percentage co-localization. Bar graphs are expressed as mean ± SEM, ***p≤0.001. (E) Sirt1-LysoTracker Red co-localization in BMDMs pretreated with Torin1 followed by S . Typhimurium infection. Sirt1-LysoTracker Red co-localization in untreated-BMDMs infected with S . Typhimurium for 4h is shown for comparison (n = 3). (F) Pearson’s correlation coefficient of Sirt1 with LysoTracker Red calculated by measuring 35 selected regions of interest (ROI) using olympus fluoview fv1000 software. (G) Immunoblot analysis of Sirt1, ACC phosphorylation and S6Kinase activation in S . Typhimurium-infected BMDMs pretreated with Torin1. (H) Mean densitometric data of Sirt1 and phosphorylated ACC were analyzed and normalized to GAPDH and total ACC respectively (n = 3). Bar graphs are expressed as mean ± SEM, ***p≤0.001 and **p≤0.01. (I) Immunoblot of phosphorylated ACC in BMDMs transfected with control or Sirt1-expressing plasmids. Western blots are representative of three experiments. (J) Densitomertic analysis of phosphorylation amounts of ACC is shown from 3 independent experiments. Bar graphs are expressed as mean ± SEM, ***p≤0.001, **p≤0.01 and *p≤0.05.

Article Snippet: Bone marrow derived macrophages (BMDMs) were prepared as described [ ] from C57BL/6J mice maintained and bred in the animal facility of Center for Molecular Medicine, University of Cologne.

Techniques: Western Blot, Infection, Phospho-proteomics, Quantitation Assay, Comparison, Software, Activation Assay, Transfection, Control, Expressing

Journal: PLoS Pathogens

Article Title: Salmonella Typhimurium disrupts Sirt1/AMPK checkpoint control of mTOR to impair autophagy

doi: 10.1371/journal.ppat.1006227

Figure Lengend Snippet: (A) Immunofluorescence image of S . Typhimurium co-localization with LC3 in GFP-LC3 expressing BMDMs at indicated time points. Data shown are representative of 3 independent experiments (n = 3). (B) Immunoblot analysis of p62 and LC3 expression upon S . Typhimurium infection in BMDMs. (C) LC3 and p62 expression levels are quantified by densitometry analysis. Data shown are from 3 independent experiments. (D) Confocal image of macrophages stained for Sirt1 and LC3. (E) BMDMs stained for LC3 and AMPK upon S . Typhimurium infection. (F) Confocal image of macrophages stained for LKB1 and LC3. (G) Immunoblot analysis of Sirt1, AMPK and LKB1 in wild type (WT) and Atg7-deficient macrophages. (H) Densitometric analysis of Sirt1, AMPK and LKB1 immunoblots (n = 3). Bar graphs are expressed as mean ± SEM, ***p≤0.001, **p≤0.01 and *p≤0.05. Scale bar = 10μm for microscopical images.

Article Snippet: Bone marrow derived macrophages (BMDMs) were prepared as described [ ] from C57BL/6J mice maintained and bred in the animal facility of Center for Molecular Medicine, University of Cologne.

Techniques: Immunofluorescence, Expressing, Western Blot, Infection, Staining

Journal: PLoS Pathogens

Article Title: Salmonella Typhimurium disrupts Sirt1/AMPK checkpoint control of mTOR to impair autophagy

doi: 10.1371/journal.ppat.1006227

Figure Lengend Snippet: Immunoblot analysis of ACC and LKB1 activation upon infection with ΔssrB (A) and ΔssaV (B) compared to S . Typhimurium. Sirt1 and acetylated-NFκB from macrophages infected with ΔssrB (C) and ΔssaV (D) compared to S . Typhimurium. (E) Expression of Sirt1 from cytoplasmic (C) and nuclear (N) fraction from BMDMs infected with ΔssrB . (F) Confocal image of Sirt1 and LysoTracker Red in ΔssrB -infected BMDMs. Sirt1-LysoTracker Red co-localization in untreated BMDMs infected with S . Typhimurium for 4h is shown for comparison (n = 3). (G) Immunoblot analysis of LC3 and p62. (H) Densitometric analysis of LC3 lipidation and p62 (n = 4). (I) Immunofluorescence image of ΔssrB -infected BMDMs stained for LC3 and LPS of S . Typhimurium (n = 3). (J) Quantitation of LC3 co-localization with SCVs. 100 SCVs were counted and expressed as percentage co-localization. (K) AKT, mTOR, p70S6K, NDRG1 expression upon S. Typhimurium (ST) and ΔssrB infection in BMDMs. (L) Densitometric analysis of AKT, mTOR, p70S6K and NDRG1 are shown from 3 independent experiments. Bar graphs are expressed as mean ± SEM, ***p≤0.001. Scale bar = 10μm for microscopical images.

Article Snippet: Bone marrow derived macrophages (BMDMs) were prepared as described [ ] from C57BL/6J mice maintained and bred in the animal facility of Center for Molecular Medicine, University of Cologne.

Techniques: Western Blot, Activation Assay, Infection, Expressing, Comparison, Immunofluorescence, Staining, Quantitation Assay

Journal: Environmental Pollution (Barking, Essex : 1987)

Article Title: Molecular mechanisms underlying NLRP3 inflammasome activation and IL-1β production in air pollution fine particulate matter (PM 2.5 )-primed macrophages ☆

doi: 10.1016/j.envpol.2023.122997

Figure Lengend Snippet: ROFA promotes the NLRP3 inflammasome-dependent release of IL-1β in murine BMDMs. (A) Confocal microscopy of BMDMs from inflammasome-reporter ASC-Citrine mice incubated with ROFA at 100 μg/mL for 6 or 24 h. White arrows indicate ASC-specks formation. LPS stimulation at 20 ng/mL for 4 h followed by the addition of 5 μM Nigericin for 2 h was used as a positive control. Time course analysis of (B) Nlrp3 , Casp1 , and Il1b mRNA expression (C) pro-IL-1β protein levels, and (D) Caspase-1 activity and IL-1β release in BMDMs from wild type (wt) mice incubated with ROFA at 100 μg/mL. (E) IL-1β release in cell culture supernatants from wt, Nlrp3 −/− , and Casp1 −/− BMDMs incubated with ROFA at 100 μg/mL for 6 or 24 h. (F) Representative dot-plots of ASC-Citrine BMDMs incubated with ROFA at 100 μg/mL for 6 or 24 h, with or without pre-incubation with MCC950. (G) Quantification of ASC-Citrine fluorescence in ASC-Citrine BMDMs incubated with ROFA at 100 μg/mL for 6 or 24 h. (H) IL-1β levels in cell culture supernatants from wt BMDMs incubated with ROFA at 100 μg/mL for 6 or 24 h, with or without pre-incubation with MCC950. Data are presented as mean ± SEM from at least three independent experiments.

Article Snippet: THP-1-ASC-GFP cells and ASC-Citrine BMDMs were acquired in a FACSCanto II equipment (BD Biosciences, Franklin Lakes, NJ, US).

Techniques: Confocal Microscopy, Incubation, Positive Control, Expressing, Activity Assay, Cell Culture, Fluorescence

Journal: Blood

Article Title: A novel inflammatory pathway mediating rapid hepcidin-independent hypoferremia

doi: 10.1182/blood-2014-08-595256

Figure Lengend Snippet: Identification of TLR6 as a novel regulator of ferroportin protein. (A) A stable and doxycycline-inducible HeLa cell line expressing a human ferroportin-Renilla luciferase fusion protein (Fpn-RLuc) was used for RNAi screening. Renilla luciferase activity (Rluc), used as a reporter of ferroportin expression, was measured 70 hours after reverse transfection of siRNA pools. The screen was performed in duplicates and the cellHTS2 software was used for data analysis. (B) Rluc activity was measured upon scramble (scr) or TLR6 interference with pooled siRNAs in the HeLa cell line expressing Fpn-Rluc and in a HeLa cell line expressing only the reporter protein. Data are presented as means ± SEM from at least 4 independent experiments. *P < .05; Student t test. (C,F) Western blot analysis of endogenous ferroportin expression in BMDMs isolated from WT or TLR6-deficient (TLR6 KO) mice or TLR2-deficient (TLR2 KO) mice; β-actin was used as loading control. Western blot images were acquired and quantified with the Vilber Lourmat Fusion-FX Chemiluminescence system. (D-E) Ferroportin and hepcidin mRNA levels were determined by qRT-PCR and calibrated to 36B4 mRNA levels. Data are means ± SEM; BMDMs were derived from at least 4 different mice per group. Each lane in the Western blot analysis represents the protein lysate obtained from a single mouse. **P < .01; Student t test.

Article Snippet: Bone marrow–derived macrophages (BMDMs) were obtained from TLR6- or TLR2-deficient mice housed at Forschungszentrum (Borstel, Germany) or at the Universitatsklinikum (Essen, Germany), respectively.

Techniques: Expressing, Luciferase, Activity Assay, Transfection, Software, Western Blot, Isolation, Quantitative RT-PCR, Derivative Assay

Journal: Blood

Article Title: A novel inflammatory pathway mediating rapid hepcidin-independent hypoferremia

doi: 10.1182/blood-2014-08-595256

Figure Lengend Snippet: FSL1-mediated TLR2/6 ligation reduces ferroportin expression in BMDMs without activating hepcidin mRNA expression. (A,C) qRT-PCR analysis of ferroportin mRNA in BMDMs from WT and TLR6-deficient mice (A), and from WT and TLR2-deficient mice (C) stimulated with FSL1 (20 ng/mL or 100 ng/mL) for the indicated time. (B,D) Western blot analysis and quantification of ferroportin expression in BMDMs from WT and TLR6-deficient mice (B) and from WT and TLR2-deficient mice (D) treated with 100 ng/mL FSL1 for 24 hours. β-actin detection ascertains equal sample loading. (E-F) Ferroportin and hepcidin mRNA expression in BMDMs after FSL1 and LPS (100 ng/mL) stimulation. mRNA levels were normalized to 36B4 mRNA levels. All data are reported as means ± SEM; BMDMs were derived from at least 4 different mice per group. Each lane in the Western blot analysis represents the protein lysate obtained from a single mouse. *P < .05; **P < .01; ***P < .001; Student t test.

Article Snippet: Bone marrow–derived macrophages (BMDMs) were obtained from TLR6- or TLR2-deficient mice housed at Forschungszentrum (Borstel, Germany) or at the Universitatsklinikum (Essen, Germany), respectively.

Techniques: Ligation, Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay

Journal: Blood

Article Title: A novel inflammatory pathway mediating rapid hepcidin-independent hypoferremia

doi: 10.1182/blood-2014-08-595256

Figure Lengend Snippet: Ferroportin downregulation is mediated by TLR2 and TLR4 ligands whereas hepcidin activation is limited to the TLR4 ligand LPS in BMDMs. (A,C-D) Ferroportin mRNA expression was determined by qRT-PCR in WT (A), TLR6-deficient (C), and TLR2-deficient (D) BMDMs stimulated with 100 ng/mL TLR2 ligands (FSL1, PAM3CSK4, PamOct2C-(VPG)4VPGKG) or TLR4 ligand (LPS) for the indicated time. (B) Western blot analysis and quantification of ferroportin expression in BMDMs from WT mice treated with 100 ng/mL LPS and PAM3CSK4 for 24 hours. β-actin was used as loading control. (E-G) Hepcidin mRNA expression was analyzed in the same samples. The mRNA quantification was calibrated to 36B4 mRNA levels. All data are reported as means ± SEM; BMDMs were derived from at least 4 different mice per group. Each lane in the Western blot analysis represents the protein lysate obtained from a single mouse. *P < .05; **P < .01; ***P < .001; ****P < .0001; Student t test.

Article Snippet: Bone marrow–derived macrophages (BMDMs) were obtained from TLR6- or TLR2-deficient mice housed at Forschungszentrum (Borstel, Germany) or at the Universitatsklinikum (Essen, Germany), respectively.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay

Journal: Infection and Immunity

Article Title: Uncovering an Important Role for YopJ in the Inhibition of Caspase-1 in Activated Macrophages and Promoting Yersinia pseudotuberculosis Virulence

doi: 10.1128/IAI.00843-15

Figure Lengend Snippet: Measurement of IL-1β in supernatants of BMDMs infected with Y. pseudotuberculosis. BMDMs from C57BL/6 mice were primed with 100 ng/ml LPS for 18 h and left uninfected or infected with strain 32777 (wild type), a yopM mutant (ΔyopM), or yopJC172AΔyopM, yopER144AΔyopM, yopHR409AΔyopM, yopTC139A ΔyopM, ΔypkA ΔyopM, or ΔyopB ΔyopM double mutant at an MOI of 30. Concentrations of IL-1β in supernatants collected at 90 min postinfection were quantified by ELISA. Data represent average values ± the standard errors of the means from three independent experiments. *, P < 0.05; **, P < 0.01; ****, P < 0.0001, as determined by one-way analysis of variance compared to results for ΔyopM strain-infected macrophages.

Article Snippet: We measured the LDH released into supernatants from infected and uninfected BMDMs using a CytoTox 96 NonRadioactive Cytotoxicity Assay kit (Promega) according to the manufacturer's instructions.

Techniques: Infection, Mutagenesis, Enzyme-linked Immunosorbent Assay

Journal: Infection and Immunity

Article Title: Uncovering an Important Role for YopJ in the Inhibition of Caspase-1 in Activated Macrophages and Promoting Yersinia pseudotuberculosis Virulence

doi: 10.1128/IAI.00843-15

Figure Lengend Snippet: Measurement of caspase-1-dependent proinflammatory cytokines in supernatants of BMDMs infected with Y. pseudotuberculosis. BMDMs from C57BL/6 mice were primed with 100 ng/ml LPS for 18 h and left uninfected or infected with the 32777, ΔyopM, yopJC172A, or yopJC172A ΔyopM strain at an MOI of 30. At 90 min postinfection, supernatants were collected, and concentrations of secreted IL-1β, IL-18, or IL-1α were measured by ELISA. Data represent average values ± the standard errors of the means from three independent experiments. ***, P < 0.001; ****, P < 0.0001, as determined by one-way analysis of variance compared to values for 32777 ΔyopM-infected macrophages.

Article Snippet: We measured the LDH released into supernatants from infected and uninfected BMDMs using a CytoTox 96 NonRadioactive Cytotoxicity Assay kit (Promega) according to the manufacturer's instructions.

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: Infection and Immunity

Article Title: Uncovering an Important Role for YopJ in the Inhibition of Caspase-1 in Activated Macrophages and Promoting Yersinia pseudotuberculosis Virulence

doi: 10.1128/IAI.00843-15

Figure Lengend Snippet: Measurement of processed caspase-1, pro-IL-1β, mature IL-1β, and pyroptosis in BMDMs infected with Y. pestis strains expressing different YopJ isoforms. LPS-primed BMDMs from C57BL/6 mice were left uninfected or infected with KIM5 ΔyopM strains expressing yopJKIM, yopJYPTB, yopJCO92, or yopJC172A at an MOI of 30. (A) Lysates were collected and analyzed at 90 min postinfection using antibodies against pro-IL-1β, cleaved caspase-1, or β-actin. Quantification of the processed caspase-1 bands showed that the signal for KIM5 yopJC172A ΔyopM was approximately 1.4 times greater than that for KIM5 ΔyopM expressing the different isoforms. Supernatants were collected at 90 min postinfection and analyzed for secreted IL-1β by ELISA (B); pyroptosis was quantified by measuring LDH release (C). Data represent average values ± the standard errors of the means from three independent experiments. By one-way analysis of variance there were no significant differences between results for the KIM5 yopJKIM ΔyopM strain and those for the other strains tested.

Article Snippet: We measured the LDH released into supernatants from infected and uninfected BMDMs using a CytoTox 96 NonRadioactive Cytotoxicity Assay kit (Promega) according to the manufacturer's instructions.

Techniques: Infection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Infection and Immunity

Article Title: Uncovering an Important Role for YopJ in the Inhibition of Caspase-1 in Activated Macrophages and Promoting Yersinia pseudotuberculosis Virulence

doi: 10.1128/IAI.00843-15

Figure Lengend Snippet: Measurement of processed caspase-1, pro-IL-1β, mature IL-1β, and pyroptosis in BMDMs infected with Y. pseudotuberculosis. LPS-primed BMDMs from C57BL/6 mice were left uninfected or infected with the 32777, ΔyopM, or yopJC172AΔyopM strain at an MOI of 30. (A) After a 90-min infection, lysates were prepared and subjected to Western blotting with antibodies recognizing pro-IL-1β, caspase-1, or β-actin. Molecular masses of corresponding proteins are shown on the right. Quantification of the processed caspase-1 bands showed that the signal for the yopJC172AΔyopM strain was 4.7 times greater than that for the ΔyopM strain. Supernatants were harvested at 90 min postinfection, and secreted IL-1β was measured by ELISA (B); pyroptosis was measured by quantification of percent LDH release (C). The data in panels B and C represent average values ± the standard errors of the means from seven independent experiments. ***, P < 0.001; ****, P < 0.0001, as determined by one-way analysis of variance compared to results for ΔyopM mutant-infected macrophages.

Article Snippet: We measured the LDH released into supernatants from infected and uninfected BMDMs using a CytoTox 96 NonRadioactive Cytotoxicity Assay kit (Promega) according to the manufacturer's instructions.

Techniques: Infection, Western Blot, Enzyme-linked Immunosorbent Assay, Mutagenesis

Journal: Infection and Immunity

Article Title: Uncovering an Important Role for YopJ in the Inhibition of Caspase-1 in Activated Macrophages and Promoting Yersinia pseudotuberculosis Virulence

doi: 10.1128/IAI.00843-15

Figure Lengend Snippet: Measurement of processed caspase-1, pro-IL-1β, mature IL-1β, and pyroptosis in BMDMs infected with Y. pseudotuberculosis or Y. pestis. LPS-primed BMDMs were left uninfected or infected with 32777, 32777 ΔyopM, KIM5, or KIM5 ΔyopM for 90 min at an MOI of 30. (A) After infection, lysates were collected, processed, and subjected to Western blotting using antibodies to pro-IL-1β, cleaved caspase-1, or β-actin. Supernatants were collected and analyzed for secreted IL-1β (B) or pyroptosis by measurement of LDH release (C). Data represent average values ± the standard errors of the means from three independent experiments. ****, P < 0.0001, as determined by one-way analysis of variance comparing values in BMDMs infected with 32777 ΔyopM versus those with 32777 infection or BMDMs infected with KIM5 ΔyopM versus those infected with KIM5.

Article Snippet: We measured the LDH released into supernatants from infected and uninfected BMDMs using a CytoTox 96 NonRadioactive Cytotoxicity Assay kit (Promega) according to the manufacturer's instructions.

Techniques: Infection, Western Blot

Journal: Infection and Immunity

Article Title: Uncovering an Important Role for YopJ in the Inhibition of Caspase-1 in Activated Macrophages and Promoting Yersinia pseudotuberculosis Virulence

doi: 10.1128/IAI.00843-15

Figure Lengend Snippet: Measurement of processed caspase-1, pro-IL-1β, mature IL-1β, and pyroptosis in BMDMs infected with Y. pestis. BMDMs from C57BL/6 mice were primed with 100 ng/ml LPS for 18 h and left uninfected or infected with KIM5, KIM5 ΔyopM, KIM5 yopJC172A, or KIM5 yopJC172AΔyopM at an MOI of 30. (A) At 90 min postinfection, lysates were collected, processed, and subjected to Western blotting using antibodies against pro-IL-1β and p10, the 10-kDa subunit of caspase-1. β-Actin was blotted as a loading control. Quantification of the processed caspase-1 bands showed that the signal for KIM5 yopJC172AΔyopM was 2.3 times greater than that for KIM5 ΔyopM. At 90 min postinfection, supernatants were collected, and secreted IL-1β was measured by ELISA (B); pyroptosis was quantified as percent LDH release (C). The data in panel B and C represent average values ± the standard error of the means from three independent experiments. **, P < 0.01; ****, P < 0.0001, as determined by one-way analysis of variance compared to results for KIM5 ΔyopM-infected macrophages.

Article Snippet: We measured the LDH released into supernatants from infected and uninfected BMDMs using a CytoTox 96 NonRadioactive Cytotoxicity Assay kit (Promega) according to the manufacturer's instructions.

Techniques: Infection, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Slit2-Mediated Metabolic Reprogramming in Bone Marrow-Derived Macrophages Enhances Antitumor Immunity

doi: 10.3389/fimmu.2021.753477

Figure Lengend Snippet: (A) Histogram of Glyco Stress test performed in BMDMs from PBS- and Slit2-treated WT and PyMT mice. The figure on the right represents kinetics of the assay, normalized to a steady reading at baseline. (B) Mito Stress test performed in BMDMs from PBS- and Slit2-treated WT and PyMT mice. The figure on the right represents kinetics of the assay, normalized to a steady reading at baseline. (C) Baseline oxygen consumption rate in response to pretreatment with BSA-conjugated free fatty acids. (D) Substrate dependence derived using the Mito Fuel Flex assay. The graph on the left represents the difference in substrate dependence between BMDMs from PBS- and Slit2-treated WT mice, while the graph on the right represents the difference in substrate dependence between BMDMs from PBS- and Slit2-treated PyMT mice. The images represent n=3–5/group. *p < 0.05, WT vs. PyMT, # p < 0.05 PBS- vs. Slit2-treated groups based on one-way ANOVA, **p < 0.05 PBS- vs. Slit2-treated groups based on unpaired t-test.

Article Snippet: Furthermore, this population was enhanced in BMDMs of Slit2-treated WT (281 ± 32.8 cells/20,000 events) and PyMT mice (141.8 ± 15.3 cells/20,000 events), thus suggesting that Slit2 modulates the polarization of macrophages toward the antitumor M1 phenotype in the bone marrow.

Techniques: Derivative Assay

Journal: Frontiers in Immunology

Article Title: Slit2-Mediated Metabolic Reprogramming in Bone Marrow-Derived Macrophages Enhances Antitumor Immunity

doi: 10.3389/fimmu.2021.753477

Figure Lengend Snippet: (A) Densitometric ratio of FASN. (B) Densitometric ratio of phosphorylated mTOR to beta-actin. (C) Densiometric ration of total mTOR to beta-actin. (D) Representative blots of Western blot analysis. (E) ECAR in response to glucose in BMDMs from WT and PyMT pretreated with mTOR inhibitor deforolimus (mTORi) and later treated with PBS or Slit2. (F) Glyco Stress test performed in BMDMs treated with Slit2 in the presence or absence of glycolysis inhibitor, galactose. (G) Baseline oxygen consumption rate in BMDMs treated with Slit2 in the presence or absence of glycolysis inhibitor, galactose. Images represent n = 3–6/group. *p < 0.05, assessed by unpaired t-test, # p < 0.05 in mTORi and PBS pretreated cells.

Article Snippet: Furthermore, this population was enhanced in BMDMs of Slit2-treated WT (281 ± 32.8 cells/20,000 events) and PyMT mice (141.8 ± 15.3 cells/20,000 events), thus suggesting that Slit2 modulates the polarization of macrophages toward the antitumor M1 phenotype in the bone marrow.

Techniques: Western Blot

Journal: Frontiers in Immunology

Article Title: Slit2-Mediated Metabolic Reprogramming in Bone Marrow-Derived Macrophages Enhances Antitumor Immunity

doi: 10.3389/fimmu.2021.753477

Figure Lengend Snippet: (A) Relative abundance of α-ketoglutarate in PBS- and Slit2-treated WT and PyMT mouse BMDMs. (B) Relative abundance of succinic acid in PBS- and Slit2-treated WT and PyMT mouse BMDMs. (C) Ratio of α-ketoglutarate:succinic acid in PBS- and Slit2-treated WT and PyMT mouse BMDMs. (D) Relative abundance of α-ketoglutarate in PBS- and Slit2-treated human BMDMs. (E) Relative abundance of succinic acid in PBS- and Slit2-treated human BMDMs. (F) Ratio of α-ketoglutarate: succinic acid in PBS- and Slit2-treated human BMDMs. Images represent n = 4–6/group. *p < 0.05, assessed by unpaired t-test.

Article Snippet: Furthermore, this population was enhanced in BMDMs of Slit2-treated WT (281 ± 32.8 cells/20,000 events) and PyMT mice (141.8 ± 15.3 cells/20,000 events), thus suggesting that Slit2 modulates the polarization of macrophages toward the antitumor M1 phenotype in the bone marrow.

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