Journal: The Journal of general virology

Article Title: Orf virus-encoded chemokine-binding protein is a potent inhibitor of inflammatory monocyte recruitment in a mouse skin model.

doi: 10.1099/vir.0.009589-0

Figure Lengend Snippet: Fig. 2. CBP–FLAG inhibits LPS-induced recruitment of Gr-1+/ CD11b+ inflammatory monocytes into the skin. Mice were injected in the dermis with LPS (1 mg) with and without CBP. In addition, each mouse received injections of CBP only and PBS i.e. a total of four injections per mouse (n53 animals per group). At 24 h post- injection, cells were isolated from skin and stained for Gr-1 and CD11b. Untreated skin was also included. Cell counts are shown for Gr-1+/CD11b+ (a), Gr-1+/CD11b” (b) and eGFP/Gr-1+/ CD11b+ (c) cells. The data shown are the mean±SD of three combined experiments i.e. n59 mice per group. Asterisks indicate results that are significantly different (P,0.01; ANOVA, paired Student’s t test).

Article Snippet: By this time, monocytes were 90 % positive for CD11b (allophycocyanin-conjugated rat anti-mouse integrin/CD11b, clone M1/70, isotype rat IgG2b; R&D Systems) and expressed low levels of MHC-II (phycoerythrinconjugated rat anti-mouse I-A/I-E, clone M5/114.15.2, isotype rat IgG2b, k; BD Biosciences) (Supplementary Fig. S1, available in JGV Online).

Techniques: Injection, Isolation, Staining

Journal: The Journal of general virology

Article Title: Orf virus-encoded chemokine-binding protein is a potent inhibitor of inflammatory monocyte recruitment in a mouse skin model.

doi: 10.1099/vir.0.009589-0

Figure Lengend Snippet: Fig. 3. Histological analyses of the effects of CBP on inflammatory cell recruitment. C57BL/6 mice were injected in the abdominal skin with 1 mg LPS with or without 100 ng CBP (n52 animals per group). Twenty-four hours later, mice were euthanized and skin sec- tions were excised and stained for various inflammatory cell types (a); these were H & E staining (leukocytes), CD11b+ (mono- cytes) and peroxidase (neutrophils). (b–d) Cells [leukocytes (b), CD11b+ monocytes (c) and neutrophils (d)] were quantified by counting in 10 high-powered fields (40 magnification). The data shown are the mean±SD.

Article Snippet: By this time, monocytes were 90 % positive for CD11b (allophycocyanin-conjugated rat anti-mouse integrin/CD11b, clone M1/70, isotype rat IgG2b; R&D Systems) and expressed low levels of MHC-II (phycoerythrinconjugated rat anti-mouse I-A/I-E, clone M5/114.15.2, isotype rat IgG2b, k; BD Biosciences) (Supplementary Fig. S1, available in JGV Online).

Techniques: Injection, Staining

Journal: Cell Death & Disease

Article Title: EGFR tyrosine kinase activity and Rab GTPases coordinate EGFR trafficking to regulate macrophage activation in sepsis

doi: 10.1038/s41419-022-05370-y

Figure Lengend Snippet: A – F Macrophages were treated with LPS (1 µg/mL) for 6, 12, or 24 h with or without PD168393 (PD, 10 µM)) pretreatment for 30 min. A Flow cytometry analysis of EGFR cell surface expression in BMDM. B Percentage of EGFR-positive macrophage is shown. C Mean fluorescence intensity (MFI) is shown ( n = 3). D Flow cytometry analysis of EGFR cell surface expression in RAW264.7. E Percentage of EGFR-positive macrophage is shown. F Mean fluorescence intensity (MFI) is shown ( n = 3). G – I Macrophages were collected from peritoneal lavage of C57BL/6 mice subjected to CLP and were divided into Sham-operated, CLP and CLP plus Erlotinib (100 mg/kg, gavage) pretreatmend for 2 h, and peritoneal macrophages were identified with CD45 + CD11b + F4/80high. G EGFR intensity on the surface of peritoneal macrophage was analyzed by flow cytometry. H Percentage of EGFR-positive peritoneal macrophage is shown ( n = 9). I Mean fluorescence intensity (MFI) is shown ( n = 9). J , K Blood was collected from patients with clinical sepsis or health donors, and blood mononuclear cells were identified with CD14 and CD11c. J EGFR intensity on the surface of Mononuclear cells were analyzed by flow cytometry (20 Healthy donors and 33 Septic patients). K Percentage of EGFR-positive CD14 + CD11c + mononuclear cell is shown. The graphs depict mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against α-HA (51064-2-AP, proteintech Group), α-FLAG (20543-1-AP, proteintech Group), APC anti-mouse CD11b (APC-65055, proteintech Group) were purchased from proteintech Group.

Techniques: Flow Cytometry, Expressing, Fluorescence

Journal: Cell Death & Disease

Article Title: EGFR tyrosine kinase activity and Rab GTPases coordinate EGFR trafficking to regulate macrophage activation in sepsis

doi: 10.1038/s41419-022-05370-y

Figure Lengend Snippet: A – F BMDMs were treated with LPS (1 μg/mL) for 24 h with or without PD168393 (10 μM) pretreatment for 30 min. A RT-qPCR analysis of mRNA expression of IL-1β ( n = 3). B RT-qPCR analysis of mRNA expression of iNOS ( n = 3). C Western blot was used to detect the expression of iNOS. D iNOS expression on the surface of BMDM was analyzed by flow cytometry. E Percentage of iNOS -positive BMDM is shown ( n = 3). F Mean fluorescence intensity (MFI) is shown ( n = 3). G – I Macrophages were collected from bronchoalveolar lavage fluid of C57BL/6 mice subjected to CLP and were divided into sham-operated, CLP and CLP plus Erlotinib (100 mg/kg, gavage) pretreatmend for 2 h, and alveolar macrophages were identified with CD45 + CD11b + F4/80high. G iNOS expression on the surface of alveolar macrophage was analyzed by flow cytometry. H Percentage of iNOS-positive alveolar macrophage is shown ( n = 9). I Mean fluorescence intensity (MFI) is shown ( n = 9). J Cluster analysis of differentially expressed metabolites in RAW264.7 measured after treated with LPS (1 μg/mL) for 30 min with or without PD168393 (PD 10 μM) pretreatment for 30 min, when compared with control. Blue and red indicates down-or upregulation, respectively ( n = 3 samples of each condition). K Schematic illustrating the metabolites that are decreased (blue) in PD168393 (10 μM) RAW264.7 cells at 24 h after LPS stimulation. L PD168393 (10 μM) pretreatment RAW264.7 cells exhibited a ~2 -fold decrease in lactate levels compared with LPS group ( n = 3). M Representative western blots of HIF1-a, p-PKM2, PKM2, LDHA expression in RAW264.7 cells. The graphs depict mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against α-HA (51064-2-AP, proteintech Group), α-FLAG (20543-1-AP, proteintech Group), APC anti-mouse CD11b (APC-65055, proteintech Group) were purchased from proteintech Group.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Flow Cytometry, Fluorescence, Control

Journal: Cell Death & Disease

Article Title: EGFR tyrosine kinase activity and Rab GTPases coordinate EGFR trafficking to regulate macrophage activation in sepsis

doi: 10.1038/s41419-022-05370-y

Figure Lengend Snippet: A – F RAW264.7 macrophages were treated with LPS (1 μg/mL) for 24 h, with or without Erlotinib (20 μM) pretreatment for 30 min. A RT-qPCR analysis of mRNA expression of M2-related genes Mcr1 ( n = 3). B RT-qPCR analysis of mRNA expression of M2-related genes Ym1 ( n = 3). C Representative western blot of Arg1. D Flow cytometry analysis showing the level of M2 macrophage-associated markers CD206. E Percentage of CD206-positive RAW264.7 is shown ( n = 3). I Mean fluorescence intensity (MFI) is shown ( n = 3). G – I Macrophages were collected from bronchoalveolar lavage fluid of C57BL/6 mice subjected to CLP and were divided into sham-operated, CLP and CLP plus Erlotinib (100 mg/kg, gavage) pretreatmend for 2 h, and alveolar macrophages were identified with CD45 + CD11b + F4/80high. G CD206 expression on the surface of alveolar macrophage was analyzed by flow cytometry. H Percentage of CD206-positive alveolar macrophage is shown ( n = 9). I Mean fluorescence intensity (MFI) is shown ( n = 9). J Immunoblot analysis of p-PPARγ (Ser112), t-PPARγ in RAW264.7 cells treated with LPS (1 μg/mL) for 30 min with or without indicated concentration of PD168393 (10 μM) pretreatment for 30 min. K Fluorescence images depicting PPARγ translocation (left panel, scale bar, 50 μm; right panel, scale bar, 5 μm). L – N RAW264.7 cells were treated with LPS (1 μg/mL) for 24 h with or without Erlotinib (10 μM) or Rosiglitazone (Rosi (20 μM) pretreatment. L Cell surface CD206 were analyzed by flow cytometry. M Percentage of CD206-positive RAW264.7 is shown ( n = 3). N Mean fluorescence intensity (MFI) is shown ( n = 3). O – Q Macrophages were collected from bronchoalveolar lavage fluid of C57BL/6 mice subjected to CLP and were divided into Sham-operated, CLP and CLP plus Erlotinib (100 mg/kg, gavage) pretreatmend for 2 h, and alveolar macrophages were identified with CD45 + CD11b + F4/80high. O CD206 expression on the surface of alveolar macrophage was analyzed by flow cytometry. P Percentage of CD206-positive alveolar macrophage is shown ( n = 9). Q Mean fluorescence intensity (MFI) is shown ( n = 9). R – V RAW264.7 cells were treated with LPS (1 μg/mL) for 30 min with or without PD168393 (10 μM) pretreatment for 30 min. R Flow cytometry analysis of JC-1 for the detecting the change of mitochondrial membrane potential (ΔΨm) (left panel, JC-1 aggregates; right panel, JC-1 monomers). S The ratio of JC-1 aggregates /JC-1 monomers was calculated as Δψm. T Total cellular ATP level was detected ( n = 3). U Immunoblot analysis of IRG1, ATP5A, SDHA, Tubulin as a loading control. V Immunoblot analysis of SDHA and IRG1 in Control or IRG1 silenced RAW264.7 cells, Tubulin as a loading control. The graphs depict mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against α-HA (51064-2-AP, proteintech Group), α-FLAG (20543-1-AP, proteintech Group), APC anti-mouse CD11b (APC-65055, proteintech Group) were purchased from proteintech Group.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Flow Cytometry, Fluorescence, Concentration Assay, Translocation Assay, Membrane, Control

Journal: Cell Death & Disease

Article Title: EGFR tyrosine kinase activity and Rab GTPases coordinate EGFR trafficking to regulate macrophage activation in sepsis

doi: 10.1038/s41419-022-05370-y

Figure Lengend Snippet: A – E Macrophages were collected from bronchoalveolar lavage fluid of C57BL/6 mice subjected to CLP and were divided into sham-operated, CLP and CLP plus Erlotinib (100 mg/kg, gavage) pretreatmend for 2 h, and alveolar macrophages were identified with CD45 + CD11b + F4/80high. A iNOS and CD206 expression on the surface of alveolar macrophage were analyzed by flow cytometry. B Percentage of iNOS-positive alveolar macrophage is shown ( n = 9). C Mean fluorescence intensity (MFI) of iNOS is shown ( n = 9). D Percentage of CD206-positive alveolar macrophage is shown ( n = 9). E Mean fluorescence intensity (MFI) of CD206 is shown ( n = 9). F – J Mice were injected intraperitoneally with LPS (20 mg/kg) or an equal volume of PBS, after pretreatment with Erlotinib (100 mg/kg) for 2 h. F The BALF neutrophils were analyzed by flow cytometry with neutrophils markers (CD11b and Ly6G). G Percentage of CD11b + Ly6G+ positive alveolar macrophage is shown ( n = 9). H Mean fluorescence intensity (MFI) of CD11b + Ly6G+ is shown ( n = 9). I Immune-staining of iNOS and CD206 in Lung sections. J Lung sections were stained with hematoxylin and eosin (H&E). Scale bar, 50 μm (upper panel) and 5 μm (lower panel). The graphs depict mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies against α-HA (51064-2-AP, proteintech Group), α-FLAG (20543-1-AP, proteintech Group), APC anti-mouse CD11b (APC-65055, proteintech Group) were purchased from proteintech Group.

Techniques: Expressing, Flow Cytometry, Fluorescence, Injection, Staining