Journal: Nature Communications

Article Title: WDR77 inhibits prion-like aggregation of MAVS to limit antiviral innate immune response

doi: 10.1038/s41467-023-40567-5

Figure Lengend Snippet: a – f PEMs from WT, Wdr77 CKO and Mavs -/- mice respectively were collected. PEMs were then stimulated for 6 h with or without VSV, SeV, poly(I:C), HSV or HT-DNA as indicated. Culture mediums were then collected and IFN-β was measured by ELISA ( a ). Ifnb1 ( b ), Ifna4 ( c ), Isg56 ( d ), Cxcl10 ( e ), Il-6 ( f ) induction were measured by qPCR (For a , IFN-β: ** p = 0.0021 **** p < 0.0001, **** p < 0.0001, ns p = 0.0790, ns p = 0.9993 in sequence; for b , Ifnb1 : all **** p < 0.0001, ns p = 0.1813, ns p = 0.8208 in sequence; for c , Ifna4 : all **** p < 0.0001, ns p = 0.9997, ns p = 0.9998 in sequence; for d , Isg56 : all **** p < 0.0001, ns p = 0.1378; For e, Cxcl10 : all **** p < 0.0001, ns p = 0.5755, ns p = 0.7986 in sequence; for f , Il-6 : ns p = 0.9872, **** p < 0.0001, ns p = 0.9932, ns p = 0.7531, ns p = 0.9849 in sequence). g BMDMs from WT, Wdr77 CKO and Mavs -/- mice respectively were collected. BMDMs were then stimulated for 6 h with or without VSV, SeV, poly(I:C), HSV or HT-DNA as indicated. Ifnb1 induction was measured by qPCR. (All **** p < 0.0001, ns p = 0.1224, ns p = 0.9878 in sequence). h PEMs from WT, Wdr77 CKO and Mavs -/- mice were collected respectively. PEMs were infected with or without SeV for 6 h. Cells were collected for subcellular fractionation, S5 fractions were subjected to native PAGE to examine IRF3 dimer. P5 fractions were subjected to SDD-AGE to examine MAVS aggregation and WCL were subjected to SDS-PAGE to examine IRF3 phosphorylation. Data are representative of two independent experiments with similar results ( h ), or three independent experiments ( a – g ) (mean ± SEM of three biological replicates). P -value was determined by two-tailed Student’s t -test. n.s. indicates no statistical significance. Source data are provided as a Source Data file.

Article Snippet: HEK293T cells, HEK293 cells, HeLa cells, Vero cells, MEF cells, PEMs (isolated from C57BL/6 mouse) and BMDMs (isolated from C57BL/6 mouse and induced by M-CSF for 7 days) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS, ExCell Bio, FSP500), penicillin (100 U/ml) and streptomycin (100 μg/ml).

Techniques: Enzyme-linked Immunosorbent Assay, Sequencing, Infection, Fractionation, Clear Native PAGE, SDS Page, Phospho-proteomics, Two Tailed Test

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