Journal: Redox Biology

Article Title: Glutathione synthesis primes monocytes metabolic and epigenetic pathway for β-glucan-trained immunity

doi: 10.1016/j.redox.2021.102206

Figure Lengend Snippet: Gclc is dispensable for β-glucan-trained immunity in vitro. (A) Integrated mechanisms involved in the glycolysis, glutaminolysis, and GSH synthesis with relevance in trained immunity (Left). Transcriptional expression of monocytes 1 day after β-glucan exposure (Right). Gene expression analysis of monocytes exposed in vitro to β-glucan was performed using previously published RNA-seq data [ , ]. Green, significant upregulation. Black dots, carbon skeleton. Overviews of the glycolysis, glutaminolysis, and glutathione synthesis pathway are shown. (B) Quantitative RT-PCR analysis of Gclc and Gclm mRNAs expression in monocytes that were trained with 5 μg/mL β-glucan for the indicated times ( n = 6). (C) Immunobloting to detect GCLC protein in Gclc −/− and Wild type BMDMs that were trained with 5 μg/mL β-glucan ( n = 3). (D) Schematic representation of in vitro trained-immunity experimental setup. (E) Reduced and oxidized glutathione intracellular levels in Gclc −/− and WT BMDMs after 24 h exposure with 5 μg/mL β-glucan and 5 days after the resting period. Results are expressed in μmole per 1 × 10 7 cells per mL. Data are presented as bars ( n = 6/group) showing individual data points from WT and Gclc −/− mice BMDMs. (F) IL-1β, IL-6 and TNFα production by β-glucan trained Gclc −/− and WT BMDMs in response to 10 ng/mL LPS restimulation on day 7 ( n = 5). (G) Gclc −/− or WT mice BMDMs were trained by β-glucan in the presence of 10 mM GSH or not, Cytokines production were analyzed by enzyme-linked immunosorbent assay (ELISA) in response to 10 ng/mL LPS restimulation on day 7 ( n = 5). BMDMs from 5 to 6 mice, in (B , E , F , G) , data represent means ± SEM. ∗p < 0.05, by one-way ANOVA/Tukey's multiple comparisons (B , E , G) ; Each Dot represents data from individual animal, ∗∗p < 0.01, by Two-tailed Student's t-test comparing WT and Gclc −/− ; ## p < 0.01 by paired Student's t-test comparing stimulated or not with β-glucan within the same genotype ( F ). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: At day 6, macrophages were restimulated with 10 ng/mL Escherichia coli lipopolysaccharide (LPS; serotype 055:B5, Sigma-Aldrich) for an additional 24 h. Mouse BMDMs (1 × 10 6 ) were plated in 96-well plates (200 μL final volume; Corning Inc., Corning, NY, USA) and stimulated with β-glucan (Invitrogen, San Diego, CA, USA) at 5 μg/mL for 24 h. Then, cells were washed and rested for 4 days in the culture medium with 10% fetal bovine serum in the presence of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF) at 20 ng/mL.

Techniques: In Vitro, Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Redox Biology

Article Title: Glutathione synthesis primes monocytes metabolic and epigenetic pathway for β-glucan-trained immunity

doi: 10.1016/j.redox.2021.102206

Figure Lengend Snippet: Myeloid-specific Gclc deletion impairs trained immunity in vivo. (A) In vivo training mouse model via two intraperitoneal (i.p.) and secondary i.p. 1 mg β-glucan injections. (B) 10 ng/mL LPS challenge for measuring serum cytokines according to (A) ( n = 5 pooled experiments). (C) In vivo training mouse model similar to (A) but with secondary C. albicans lethal dose (∼ 2.5 × 10 6 ) infection. (D) Survival curve, according to (C) . (E) In vivo training mouse model via non-lethal C. albicans (∼ 2 × 10 4 ) training and secondary C. albicans lethal (∼ 2 × 10 6 ) infection. (F) Survival curves of WT and Gclc −/− mice trained with non-lethal C. albicans followed with a lethal C. albican infection, according to (E) . (G) The production of cytokines was determined in BMDMs s from trained WT and Gclc −/− mice, according to (E) . (H) Kidney fungal burden was determined from trained WT and Gclc −/− mice, according to (E) . In ( B , G , H ), single dots correspond to individual mouse, means ± SEM of 2 or 3 pooled experiments are shown. ∗∗p < 0.01 by Two-tailed Student's t -test comparing WT and Gclc −/− (B) ; ∗p < 0.05, ∗∗p < 0.01 by one-way ANOVA/Tukey's multiple comparisons test (bottom) (G , H) ; Pooled data of two experiments are shown, including 7–12 mice per group as indicated, ∗∗p < 0.01 by log-rank test (D , F) .

Article Snippet: At day 6, macrophages were restimulated with 10 ng/mL Escherichia coli lipopolysaccharide (LPS; serotype 055:B5, Sigma-Aldrich) for an additional 24 h. Mouse BMDMs (1 × 10 6 ) were plated in 96-well plates (200 μL final volume; Corning Inc., Corning, NY, USA) and stimulated with β-glucan (Invitrogen, San Diego, CA, USA) at 5 μg/mL for 24 h. Then, cells were washed and rested for 4 days in the culture medium with 10% fetal bovine serum in the presence of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF) at 20 ng/mL.

Techniques: In Vivo, Infection, Two Tailed Test

Journal: Redox Biology

Article Title: Glutathione synthesis primes monocytes metabolic and epigenetic pathway for β-glucan-trained immunity

doi: 10.1016/j.redox.2021.102206

Figure Lengend Snippet: Gclc deletion impairs glycolysis and glutaminolysis in trained immunity via c-Myc suppression. (A) Level of ATP in Gclc −/− and WT BMDMs that were trained with 5 μg/mL β-glucan for 24 h on day 6, without IFN-γ priming and LPS restimulation ( n = 9). (B and C) Extracellular glucose and lactate secretion rates (B) , and the ratio of molecules of lactate produced per molecule of glucose consumed (C) , in Gclc −/− and WT cells trained with 5 μg/mL β-glucan for 24 h on day 6, without IFN-γ priming and LPS restimulation ( n = 8). (D) Fluxmap of mammalian carbon metabolism (left) and mass isotopomer distribution of citrate (right) in Gclc −/− and WT BMDMs that were incubated with U- 13 C-glucose and trained with 5 μg/mL β-glucan for 24 h on day 6, without IFN-γ priming and LPS restimulation ( n = 6). (E) Determination of glutamine anaplerosis in Gclc −/− and WT BMDMs that were stimulated for 24 h with 5 μg/mL β-glucan ( n = 5). (F) Fluxmap of mammalian carbon metabolism (left) and mass isotopomer distribution of citrate (right) for Gclc −/− and WT cells that were incubated with U- 13 C-glutamine and trained with 5 μg/mL β-glucan for 24 h ( n = 6). (G) Flow-cytometric analysis of c-Myc expression in 5 μg/mL β-glucan-trained WT and Gclc −/− monocytes on day 1 ( n = 3). (H) Gclc −/− BMDMs transduced with c-Myc were trained by 5 μg/mL β-glucan for 24 h in the presence of 100 nMol/L Rapamycin (Rapa) or not, Glutamine and glucose uptake were analyzed ( n = 5). In (A to H) , data represent means ± SEM, ∗p < 0.05 by one-way ANOVA/Tukey's multiple comparisons (E , F) .

Article Snippet: At day 6, macrophages were restimulated with 10 ng/mL Escherichia coli lipopolysaccharide (LPS; serotype 055:B5, Sigma-Aldrich) for an additional 24 h. Mouse BMDMs (1 × 10 6 ) were plated in 96-well plates (200 μL final volume; Corning Inc., Corning, NY, USA) and stimulated with β-glucan (Invitrogen, San Diego, CA, USA) at 5 μg/mL for 24 h. Then, cells were washed and rested for 4 days in the culture medium with 10% fetal bovine serum in the presence of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF) at 20 ng/mL.

Techniques: Produced, Incubation, Expressing, Transduction

Journal: Redox Biology

Article Title: Glutathione synthesis primes monocytes metabolic and epigenetic pathway for β-glucan-trained immunity

doi: 10.1016/j.redox.2021.102206

Figure Lengend Snippet: Extensive ROS production upon Gclc deletion inhibits mTOR-mediated immunometabolic changes in trained immunity. (A) Flow-cytometric determination of ROS in Gclc −/− and WT BMDMs that were trained with 5 μg/mL β-glucan for 24 h and stained with DCF-DA ( n = 8). (B and C) Intracellular flow-cytometric determination of phosphorylated mTOR (B) and phospho-S6 (C) in Gclc −/− (O) and WT (W) BMDMs that were trained with 5 μg/mL β-glucan for 24 h in the presence of 10 mM NAC, or 20 mM GSH ( n = 8). (D) Immunoblot showing phospho-4EBP1 and c-Myc in Gclc −/− and WT BMDMs that were trained with 5 μg/mL β-glucan for 24 h in the presence or absence of 10 mM NAC. Loading control: actin ( n = 3). (E and F) Measurement of glucose and lactate secretion rates (E) , and the ratio of molecules of lactate produced per molecule of glucose consumed (F) , in Gclc −/− and WT BMDMs that were trained with 5 μg/mL β-glucan for 24 h in the presence or absence of 10 mM NAC ( n = 7). (G) Mass isotopomer distribution of fumarate in Gclc −/− and WT BMDMs that were trained with 5 μg/mL β-glucan for 24 h in the presence or absence of 10 mM NAC or 20 mM GSH ( n = 6). (H) Flow-cytometric determination of ROS in BMDMs that were trained with 5 μg/mL β-glucan for 24 h in the presence of 10 mM NAC ( n = 6). (I) Cytokines production in the supernatants of 5 μg/mL β-glucan-trained Gclc −/− and WT BMDMs in the presence of 10 mM NAC ( n = 6). (J) Determination of intracellular ATP concentrations in Gclc −/− and WT BMDMs that were trained with 5 μg/mL β-glucan for 24 h in the presence of 20 mM GSH, 10 mM NAC and/or 100 nM Rapamycin (Rapa) ( n = 5). (K) IL-1β production in the supernatants of 5 μg/mL β-glucan-trained Gclc −/− and WT BMDMs in response to LPS on day 7, according to (J) ( n = 7). In (A to C , E to K) , data represent means ± SEM. ∗p < 0.05 by Two-tailed Student's t-test (A-C , E-F) ; ∗p < 0.05 by one-way ANOVA/Tukey's multiple comparisons test (H-K) .

Article Snippet: At day 6, macrophages were restimulated with 10 ng/mL Escherichia coli lipopolysaccharide (LPS; serotype 055:B5, Sigma-Aldrich) for an additional 24 h. Mouse BMDMs (1 × 10 6 ) were plated in 96-well plates (200 μL final volume; Corning Inc., Corning, NY, USA) and stimulated with β-glucan (Invitrogen, San Diego, CA, USA) at 5 μg/mL for 24 h. Then, cells were washed and rested for 4 days in the culture medium with 10% fetal bovine serum in the presence of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF) at 20 ng/mL.

Techniques: Staining, Western Blot, Produced, Two Tailed Test

Journal: Redox Biology

Article Title: Glutathione synthesis primes monocytes metabolic and epigenetic pathway for β-glucan-trained immunity

doi: 10.1016/j.redox.2021.102206

Figure Lengend Snippet: Gclc deletion induces H3K4 trimethylation and H3k27me3 demethylation in trained immunity. (A and B) Ch-IP analysis of acetylate histone H3 (lysines 3 and 9, AcH3) (A) , and acetylated histone H4 (lysines 5, 8, 12, and 16, AcH4) (B) in the promoters of Gls , Glut1 , Hk2 , and Pfkp in Gclc −/− and WT BMDMs that were trained with 5 μg/mL β-glucan for 24 h on day 6, without IFN-γ priming and LPS restimulation ( n = 5). (C) Recruitment of c-Myc to the promoters of Gls , Glut1 , Hk2 in Gclc −/− and WT BMDMs that were trained with 5 μg/mL β-glucan for 24 h in the presence or absence of the HDAC inhibitor TSA (100 nM) ( n = 6). (D) Abundance of histone H3, H3K4me3, H3K27me3, and H4K20me3 in the promoters of Gls , Hk2 , and Glut1 in WT BMDMs in basal (unstimulated) conditions ( n = 6). (E) Ch-IP qPCR analysis of H3, H3K4me3, H3K27me3 in the promoters of Gls , Glut1 , Hk2 , and Pfkp in Gclc −/− and WT BMDMs that were trained with 5 μg/mL β-glucan for 24 h ( n = 6). (F) H4K20me3 enrichment in the promoters of Gls , Glut1 , Hk2 in Gclc −/− and WT BMDMs that were trained with 5 μg/mL β-glucan for 24 h ( n = 6). In (A to F) , data represent means ± SEM. ∗p < 0.05 by Two-tailed Student's t-test (A to D) ; # p < 0.05 or & p < 0.05 between untrained and β-glucan-trained WT ( # ) or Gclc −/− ( & ) monocytes, ∗p < 0.05, ∗ ∗p < 0.01 by one-way ANOVA/Tukey's multiple comparisons test (E) .

Article Snippet: At day 6, macrophages were restimulated with 10 ng/mL Escherichia coli lipopolysaccharide (LPS; serotype 055:B5, Sigma-Aldrich) for an additional 24 h. Mouse BMDMs (1 × 10 6 ) were plated in 96-well plates (200 μL final volume; Corning Inc., Corning, NY, USA) and stimulated with β-glucan (Invitrogen, San Diego, CA, USA) at 5 μg/mL for 24 h. Then, cells were washed and rested for 4 days in the culture medium with 10% fetal bovine serum in the presence of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF) at 20 ng/mL.

Techniques: Two Tailed Test

Journal: Redox Biology

Article Title: Glutathione synthesis primes monocytes metabolic and epigenetic pathway for β-glucan-trained immunity

doi: 10.1016/j.redox.2021.102206

Figure Lengend Snippet: Gclc deletion induces EZH2-mediated H3K27 trimethylation and KMD5-induced H3K4me3 demethylation in trained immunity. (A) Integrated mechanisms involved in the synthesis of GSH with relevance in epigenetic pathways. (B) Methyltransferase activity of H3K4, H4K20, and H3K27 in Gclc −/− and WT BMDMs that were trained with 5 μg/mL β-glucan for 24 h on day 6 before LPS restimulation ( n = 6). (C) Demethylase activity of KDM1, KDM5, or KDM6 in Gclc −/− and WT BMDMs that were trained with 5 μg/mL β-glucan for 24 h on day 6 before LPS restimulation ( n = 6). (D) Activity of H3K27me3 methyltransferase or KDM6 demethylase in Gclc −/− and WT BMDMs that were trained with 5 μg/mL β-glucan for 24 h in the presence or absence of 10 mM NAC ( n = 6). (E) Ch-IP qPCR analysis of H3K27me3 in the promoters of Gls , Glut1 , and Hk2 in Gclc −/− and WT BMDMs that were trained with 5 μg/mL β-glucan for 24 h in the presence or absence of 5.0 μmol/L EZH2 inhibitor (Tazverik), or 5.0 μmol/L KDM6 inhibitor GSK-J4 ( n = 6). (F) Induction of GLS, HK2, and GLUT1 mRNA in Gclc −/− and WT BMDMs that were trained with 5 μg/mL β-glucan for 24 h in the presence or absence of 5.0 μmol/L EZH2 inhibitor (Tazverik), or 5.0 μmol/L KDM6 inhibitor GSK-J4 ( n = 6). (G) Recruitment of c-Myc to the promoters of Gls , Glut1 , and Hk2 promoters in Gclc −/− and WT BMDMs that were trained with 5 μg/mL β-glucan for 24 h in the presence or absence of 5.0 μmol/L EZH2 inhibitor (Tazverik), or 5.0 μmol/L KDM6 inhibitor GSK-J4 ( n = 6). In (B to G) , data represent means ± SEM. ∗p < 0.05 by one-way ANOVA/Tukey's multiple comparisons test.

Article Snippet: At day 6, macrophages were restimulated with 10 ng/mL Escherichia coli lipopolysaccharide (LPS; serotype 055:B5, Sigma-Aldrich) for an additional 24 h. Mouse BMDMs (1 × 10 6 ) were plated in 96-well plates (200 μL final volume; Corning Inc., Corning, NY, USA) and stimulated with β-glucan (Invitrogen, San Diego, CA, USA) at 5 μg/mL for 24 h. Then, cells were washed and rested for 4 days in the culture medium with 10% fetal bovine serum in the presence of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF) at 20 ng/mL.

Techniques: Activity Assay

Journal: Redox Biology

Article Title: Glutathione synthesis primes monocytes metabolic and epigenetic pathway for β-glucan-trained immunity

doi: 10.1016/j.redox.2021.102206

Figure Lengend Snippet: Pharmacological inhibition of EZH2 rescued trained immunity impaired by Gclc deficiency. (A) In vitro experimental model applied to mouse BMDMs, indicating when the time of EZH2 inhibitor (EZH2i) Tazverik (5.0 μmol/L) treatment. (B) Mouse BMDMs were incubated with the EZH2i at the indicated concentrations. TNFα and IL-1β level was analyzed in the supernatants of β-glucan-trained cells after LPS stimulation, according to (A) . Mean ± SEM of 3 independent experiments is shown. *p < 0.05, **p < 0.01 or ***p < 0.001 by one-way ANOVA/Tukey's multiple comparisons test. (C) In vivo model of training by a systemic infection with a low dose of C. albicans in the presence of 100 mg/Kg EZH2i, followed by challenge with a lethal dose of the same pathogen. When indicated, the inhibitor was administered intraperitoneally. (D) Survival curve of control or EZH2i-treated Gclc −/− and WT mice, according to (C) . A pool of data from two experiments is shown, including between 10 and 12 mice per group as indicated. **p < 0.01, log rank test between trained control and EZH2i-treated groups. (E) In vitro experimental model applied to human peripheral blood mononuclear cells (PBMCs) in the presence of 200 μmol/L BSO indicating when 10.0 μmol/L EZH2i were added. (F) IL-1β, IL-6, and TNFα production was analyzed in supernatants of β-glucan-trained human monocytes after LPS stimulation, according to (E) . Data from 5 to 6 independent donors are shown. Dots represent data from individual samples, ∗p < 0.05, by Two-tailed Student's t-test comparing PBS and EZH2i treatment - ; # p < 0.05 by paired Student's t-test comparing β-glucan stimulation and controls within the same genotype ( F ).

Article Snippet: At day 6, macrophages were restimulated with 10 ng/mL Escherichia coli lipopolysaccharide (LPS; serotype 055:B5, Sigma-Aldrich) for an additional 24 h. Mouse BMDMs (1 × 10 6 ) were plated in 96-well plates (200 μL final volume; Corning Inc., Corning, NY, USA) and stimulated with β-glucan (Invitrogen, San Diego, CA, USA) at 5 μg/mL for 24 h. Then, cells were washed and rested for 4 days in the culture medium with 10% fetal bovine serum in the presence of recombinant granulocyte-macrophage colony stimulating factor (GM-CSF) at 20 ng/mL.

Techniques: Inhibition, In Vitro, Incubation, In Vivo, Infection, Two Tailed Test

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: 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: 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