Journal: Current Issues in Molecular Biology

Article Title: Transcriptomic Establishment of Pig Macrophage Polarization Signatures

doi: 10.3390/cimb45030151

Figure Lengend Snippet: Brief methods of bone marrow-derived macrophage induction.

Article Snippet: M2_IL4 + IL10 , M2 , 10 ng/mL IL10(Proteintech, USA, HZ-1145), and 10 ng/mL IL4 (Proteintech, Rosemount, IL, USA, HZ-1004).

Techniques:

Journal: Current Issues in Molecular Biology

Article Title: Transcriptomic Establishment of Pig Macrophage Polarization Signatures

doi: 10.3390/cimb45030151

Figure Lengend Snippet: Transcriptomic comparison of porcine macrophage phenotypes, M1 (M1_IFNγ + LPS, and M1_GM-CSF) versus M2 (M1_IFNγ + LPS, and M1_GM-CSF). ( A ) PCA clustering M1_IFNγ + LPS ( n = 4), M1_GM-CSF ( n = 4), M2_IL4 + IL10 ( n = 3), M2_M-CSF ( n = 3); ( B ) Top enriched GO biological processes of 730 DEG genes, M1 (M1_IFNγ + LPS, and M1_GM-CSF) and M2 (M2_IL4 + IL10, and M2_M-CSF); ( C ) Heatmap exhibiting 49 classical macrophage marker genes expression profile among M1_IFNγ + LPS, M1_GM-CSF, M2_IL4 + IL10, and M2_M-CSF; ( D ) The DEGs between M1 and M2 were studied by using the String online tool. The interaction between each protein pair is represented by lines, and the size of the circle is directly proportional to the degree.

Article Snippet: M2_IL4 + IL10 , M2 , 10 ng/mL IL10(Proteintech, USA, HZ-1145), and 10 ng/mL IL4 (Proteintech, Rosemount, IL, USA, HZ-1004).

Techniques: Comparison, Marker, Expressing

Journal: Current Issues in Molecular Biology

Article Title: Transcriptomic Establishment of Pig Macrophage Polarization Signatures

doi: 10.3390/cimb45030151

Figure Lengend Snippet: Comparison of transcriptomics in different phenotypes of porcine macrophages. ( A ) Heatmap showing that specific macrophage phenotypes were aggregated by the induction method, M1_IFNγ + LPS, and M1_GM-CSF; ( B ) Heatmap showing that specific macrophage phenotypes were aggregated by the induction method M2_IL4 + IL10 and M2_M-CSF; ( C ) GO pathway enrichment analysis of two polarized macrophages, M1_IFNγ + LPS and M1_GM-CSF; ( D ) GO pathway enrichment analysis of two polarized macrophages, M2_IL4 + IL10 and M2_M-CSF; ( E ) The DEGs were studied by using the String online tool, M1_IFNγ + LPS, and M1_GM-CSF. The interaction between each protein pair is represented by lines, and the size of the circle is directly proportional to the degree of interaction. We adopt a node number greater than 1; ( F ) The DEGs are studied by using the String online tool, M2_IL4 + IL10, and M2_M-CSF. The interaction between each protein pair is represented by lines, and the size of the circle is directly proportional to the degree of interaction. We adopt a node number greater than 2.

Article Snippet: M2_IL4 + IL10 , M2 , 10 ng/mL IL10(Proteintech, USA, HZ-1145), and 10 ng/mL IL4 (Proteintech, Rosemount, IL, USA, HZ-1004).

Techniques: Comparison

Journal: Current Issues in Molecular Biology

Article Title: Transcriptomic Establishment of Pig Macrophage Polarization Signatures

doi: 10.3390/cimb45030151

Figure Lengend Snippet: Validation of M1_IFNγ + LPS, M1_GMCSF, M2_M-CSF, and M2_IL4 + IL10 signatures. ( A ) SS2-infected macrophages were enriched in M1_IFNγ + LPS based on GSEA (Data source: Microarray analysis); ( B ) PRRSV-infected macrophages were enriched in M1_GM-CSF based on GSEA (Data source: RNA-seq); ( C ). T. gondii -infected macrophages were enriched in M2_IL4 + IL10 based on GSEA (Data source: RNA-seq); ( D ) T. gondii Me49 -infected macrophages were enriched in M2_M-CSF based on GSEA (Data source: RNA-seq).

Article Snippet: M2_IL4 + IL10 , M2 , 10 ng/mL IL10(Proteintech, USA, HZ-1145), and 10 ng/mL IL4 (Proteintech, Rosemount, IL, USA, HZ-1004).

Techniques: Biomarker Discovery, Infection, Microarray, RNA Sequencing

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: Killing efficiency of different physicochemical treatments on recombinant L. lactis strains displaying different binders of proinflammatory cytokines on their surface. The killing efficiency was determined by culturing and counting colonies on agar plates. Complete killing of bacteria i.e., 0 CFU/mL (no bacterial colonies on agar plates) was considered as 100% efficiency. pSD-ZIL6, L. lactis displaying anti-IL6 affibody; pSD-ZTNF, L. lactis displaying anti-TNF affibody; pSD-Fyn17, L. lactis displaying anti-IL17 fynomer; pSD-EVA, L. lactis displaying anti-IL8 evasin; pNZ8148, L. lactis harboring empty plasmid. The results presented are means ± standard deviation (SD) of three technical replicates from a representative experiment.

Article Snippet: Briefly, serial dilutions of IL6 (0.32, 0.65, 1.31, 2.62, 5.25, 10.5, 21, 42 nM; HZ-1019, Proteintech) were incubated with a fixed number of recombinant bacteria (8 × 10 6 CFU) for 2 h in a final volume of 200 μl.

Techniques: Recombinant, Bacteria, Plasmid Preparation, Standard Deviation

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: Cytokine binding ability of recombinant L. lactis bacteria displaying different binders of proinflammatory cytokines on their surface upon exposure to bacteria-killing treatments. ELISA-determined concentrations of recombinant IL6 (A) , TNF (B) , IL17 (C) , and IL8 (D) that remained in the solution following incubation with the corresponding strain of bacteria before and after treatment with heat (70 °C, 40 min or 100 °C, 30 min), sonication, ethanol, UV, and gamma irradiation. pSD-ZIL6, L. lactis displaying ZIL6 affibody; pSD-ZTNF, L. lactis displaying anti-TNF affibody; pSD-Fyn17, L. lactis displaying anti-IL17 fynomer; pSD-EVA, L. lactis displaying anti-IL8 evasin; Ctrl: L. lactis control cells containing empty plasmid pNZ8148. The results are presented as means ± SD of three technical replicates of a representative experiment. ns, p = 0.09; *, p ≤ 0.05; **, p = 0.002; ***, p < 0.001 (unpaired two-tailed t -test).

Article Snippet: Briefly, serial dilutions of IL6 (0.32, 0.65, 1.31, 2.62, 5.25, 10.5, 21, 42 nM; HZ-1019, Proteintech) were incubated with a fixed number of recombinant bacteria (8 × 10 6 CFU) for 2 h in a final volume of 200 μl.

Techniques: Binding Assay, Recombinant, Bacteria, Enzyme-linked Immunosorbent Assay, Incubation, Sonication, Irradiation, Control, Plasmid Preparation, Two Tailed Test

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: Determination of binding affinity of ZIL6-displaying L. lactis for human IL6. A constant number of 8 × 10 6 CFU bacteria was incubated with increasing concentrations of human IL6 for 2 h. MFI values were measured by flow cytometry and binding curves were fitted to the data using the Hill equation in the GraphPad Prism v.10.3.1. The dissociation constants (Kd) values were calculated from the binding curves for non-treated, live ZIL6-displaying L. lactis bacteria and bacteria treated by heat (70 °C, 40 min or 100 °C, 30 min), sonication, ethanol, and UV irradiation. Data are means ± SD of three technical replicates from a representative experiment.

Article Snippet: Briefly, serial dilutions of IL6 (0.32, 0.65, 1.31, 2.62, 5.25, 10.5, 21, 42 nM; HZ-1019, Proteintech) were incubated with a fixed number of recombinant bacteria (8 × 10 6 CFU) for 2 h in a final volume of 200 μl.

Techniques: Binding Assay, Bacteria, Incubation, Flow Cytometry, Sonication, Irradiation

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: Quantification of the maximum binding capacity of ZIL6-displaying L. lactis . Increasing concentrations of recombinant human IL6 (0.45, 4.5, 45 and 450 ng) were incubated with a constant number of bacteria (4.5 × 10 7 CFU equivalent to 0.1 mg dry cell weight) in 450 μl. Residual IL6 in the supernatants was quantified by ELISA for live non-treated bacteria and the cells treated by heat (70 °C, 40 min or 100 °C, 30 min), sonication, ethanol, and UV irradiation. The percentage of bound IL6 was calculated from the difference measured in the presence of ZIL6-displaying L. lactis and control bacteria (carrying empty plasmid pNZ8148). The data are means ± SD of two biological replicates. The asterisks denote statistically significant differences between treated and non-treated bacteria for each concentration. ***, p < 0.001 (one-way ANOVA with Dunnett multiple comparison test).

Article Snippet: Briefly, serial dilutions of IL6 (0.32, 0.65, 1.31, 2.62, 5.25, 10.5, 21, 42 nM; HZ-1019, Proteintech) were incubated with a fixed number of recombinant bacteria (8 × 10 6 CFU) for 2 h in a final volume of 200 μl.

Techniques: Binding Assay, Recombinant, Incubation, Bacteria, Enzyme-linked Immunosorbent Assay, Sonication, Irradiation, Control, Plasmid Preparation, Concentration Assay, Comparison

Journal: Frontiers in Microbiology

Article Title: Postbiotics derived from recombinant lactic acid bacteria exhibit high IL6-binding capacity and suppress IL6-induced STAT3 signaling

doi: 10.3389/fmicb.2025.1657810

Figure Lengend Snippet: (A) Inhibition of STAT3 signaling in HEKblue-IL6R cells by non-viable ZIL6-displaying L. lactis bacteria in comparison to live non-treated strain. ZIL6-displaying L. lactis cells (1 × 10 8 CFU/mL or 1 × 10 9 CFU/mL) were preincubated with IL6 (1 ng/mL) for 2 h. After removal of bacterial cells, the cell-free supernatant containing remainder of IL6 was added to HEKblue-IL6R cells to induce the reporter system. The percentage of STAT3 inhibition was calculated relative to the STAT3 signaling induced by IL6 in the absence of bacteria. The inhibition of STAT3 signaling was determined for live non-treated bacteria and the cells treated by heat (70 °C, 40 min or 100 °C, 30 min), sonication, ethanol, UV, and gamma irradiation. pSD-ZIL6, L. lactis displaying ZIL6 affibody; Ctrl: L. lactis control cells containing empty plasmid pNZ8148. An anti-IL6 monoclonal antibody (Ab) was used as a positive control. The data are means ± SD of four biological replicates. The asterisks denote statistically significant differences between treated and non-treated bacteria for each concentration. ***, p < 0.001 (one-way ANOVA with Dunnett multiple comparison test). (B) The effect of lactic acid production by L. lactis on viability of HEKblue-IL6R cells. HEKblue-IL6R cells (100 000 cells/well) were incubated with ZIL6-displaying L. lactis (2 × 10 7 bacteria/well) for 6, 12, and 24 h, and the viability of HEKblue-IL6R cells was determined with trypan blue. The data are means ± SD of three technical replicates of a representative experiment.

Article Snippet: Briefly, serial dilutions of IL6 (0.32, 0.65, 1.31, 2.62, 5.25, 10.5, 21, 42 nM; HZ-1019, Proteintech) were incubated with a fixed number of recombinant bacteria (8 × 10 6 CFU) for 2 h in a final volume of 200 μl.

Techniques: Inhibition, Bacteria, Comparison, Sonication, Irradiation, Control, Plasmid Preparation, Positive Control, Concentration Assay, Incubation

Journal: The Journal of Neuroscience

Article Title: Functional Recovery after Peripheral Nerve Injury is Dependent on the Pro-Inflammatory Cytokines IL-1β and TNF: Implications for Neuropathic Pain

doi: 10.1523/JNEUROSCI.2840-11.2011

Figure Lengend Snippet: Summary of the primary antibodies used in the present study

Article Snippet: The complete description of the primary antibodies used in this study is given in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Specificity Immunogen/clone Dilution Source Catalog/lot # Flow cytometry 7/4 (recently identified as Ly-6B), PE-conjugated 25–30 kDa GPI-anchored protein expressed in neutrophils and proinflammatory M1 monocytes Clone 7/4 1:100 AbD Serotec MCA771PE CD11b (Integrin αM chain, Mac-1 α chain), BD Horizon V450-conjugated Murine αM chain of Mac-1 found on myeloid cells Clone M1/70 1:333 BD Biosciences 560455/65504 CD45 (Leukocyte Common Antigen, Ly-5), PerCP-conjugated CD45 molecule on all leukocytes Clone 30-F11 1:167 BD Biosciences 557235/51129 Ly-6C, FITC-conjugated 14–17 kDa GPI-anchored protein expressed at various levels in monocyte/macrophage subsets, granulocytes, and T cell subsets Clone AL-21 1:500 BD Biosciences 553104/41637 Ly-6G, PE-Cy7-conjugated 21–25 kDa GPI-anchored protein expressed in neutrophils Ly-6G-transfected EL4J cell line Clone 1A8 1:500 BD Biosciences 560601/44917 Immunofluorescence labeling 7/4 25–30 kDa GPI-anchored protein expressed in neutrophils and proinflammatory M1 monocytes Clone 7/4 1:800 AbD Serotec MCA771GA Ionized calcium-binding adaptor molecule 1 (iba1) 17 kDa EF hand protein expressed in macrophages and microglia Synthetic peptide of C-terminus iba1 N′-PTGPPAKKAISELP-C′ 1:750 Wako 019-19741/CDF1873 Ly-6G 21–25 kDa GPI-anchored protein expressed in neutrophils Ly-6G-transfected EL4J cell line Clone 1A8 1:5000 BD Biosciences 551459/26029 Immunoblotting β-Actin Antibody reacts with all vertebrate actin isoforms Purified chicken gizzard actin Clone C4 1:7000 Millipore (Chemicon) MAB1501 Caspase-1 Antibody reacts with full-length caspase-1 and cleaved forms that retain aa 371–390 Synthetic peptide of amino acids 371–390 of caspase-1 (human) Clone 14F468 1:300 Imgenex IMG-5028/AB093004A-3 Interleukin-1 α (IL-1α) Based on amino acid composition, the theoretical MW of the recognized protein is 31 kDa Highly pure (>98%) recombinant mouse IL-1α 1:1250 Abcam ab9724 Interleukin-1 β (IL-1β) 17 kDa processed form of IL-1β Synthetic peptide of N-terminal region of mature IL-1β (human) 1:1000 Cell Signaling 2021 Tumor necrosis factor α (TNF) 26 kDa membrane-bound TNF precursor (pro-TNF) Recombinant full-length TNF protein (human) 1:1250 Abcam ab6671 TNF-α converting enzyme (TACE) Antibody reacts with mature, precursor and glycosylated forms of TACE (also termed Adam17) Synthetic peptide of C-terminus TACE (human) 1:300 Lab Vision (NeoMarkers) RB-1660-P/1660P807A Open in a separate window Summary of the primary antibodies used in the present study

Techniques: Flow Cytometry, Immunofluorescence, Labeling, Western Blot, Purification, Recombinant, Membrane