Journal: Nature communications

Article Title: Primate-Specific miR-576-3p Sets Host Defense Signaling Threshold

doi: 10.1038/ncomms5963

Figure Lengend Snippet: a , Schematic showing the region of the 3’ UTR of STING that is recognized by the miR-576-3p seed sequence. b , HBEC were transfected with control or miR-576-3p (M-576-3p) mimic for 72 h. Cells were then mock infected or infected with VSV-GFP at an MOI 3 for 3 h. Total RNA was harvested from cells and STING mRNA levels were determined by qPCR and normalized to levels of β-actin. c , HBEC were transfected with miRNA mimic (M-576-3p) or inhibitor (I-576-3p) as in b and mock-infected or infected with VSV-GFP at MOI 0.1 for 18 h. Cell lysates were harvested and subjected to western blot analysis with anti-STING antibodies. β-actin serves as loading control. d,e , HBEC were transfected with 1 µg/ml of poly (I:C) for 6 h ( d ) or treated with 100 U/ml of IFNβ for 18 h ( e ) and levels of pri-mir-576 and SEC24B mRNA were analyzed by qPCR. f,g , HBEC were transfected with poly (I:C) as in d after siRNA knockdown of NFκB (P65) or IRF3. Relative pri-mir-576 ( f ) or SEC24B mRNA ( g ) levels were measured by qPCR. h , HBEC expressing control or miR-576-3p mimic (M-576-3p) or an siRNA targeting STING were transfected with poly (I:C) and levels of IFNβ mRNA were measured by qPCR. i , HBEC were transfected with control or miR-576-3p mimic (M-576-3p) and pre-treated with 100 U/ml of IFNβ prior to VSV infection. Cell viability was determined by measuring ATP levels of mock or infected cells. j , HBEC expressing control or miR-576-3p inhibitor (I-576-3p) were transfected with poly (I:C) and levels of IFNβ were measured by qPCR. Data are representative of three independent experiments. Unpaired two tailed t-test was used and error bars represent SD. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Recombinant human IFNβ was obtained from R&D systems.

Techniques: Sequencing, Transfection, Control, Infection, Western Blot, Knockdown, Expressing, Two Tailed Test

Journal: Nature communications

Article Title: Primate-Specific miR-576-3p Sets Host Defense Signaling Threshold

doi: 10.1038/ncomms5963

Figure Lengend Snippet: a , HBEC were transfected with 1 µg/ml of poly (I:C) for 6 h or treated with 100 U/ml of IFNβ for 18 h and levels of mature miR-576-3p were determined by qPCR. b , HBEC were mock infected or infected with HSV-1 at MOI 10 for 3 h and levels of miR-576-3p were measured by qPCR. Unpaired two tailed t-test was used and error bars represent SD. *p<0.05, **p<0.01. ns, non significant with respect to control.

Article Snippet: Recombinant human IFNβ was obtained from R&D systems.

Techniques: Transfection, Infection, Two Tailed Test, Control

Journal: Nature communications

Article Title: Primate-Specific miR-576-3p Sets Host Defense Signaling Threshold

doi: 10.1038/ncomms5963

Figure Lengend Snippet: a , Model illustrates regulation of IFN expression by miR-576-3p as a feedback mechanism. Targets of miR-576-3p are indicated by inhibitory red lines. b , Public available microRNA array datasets (GSE37425 and GSE37426) of synovial or renal tissues from RA or SLE patients, respectively, along with controls were obtained from the GEO database . Normalized expression values of miR-576-3p from each sample were used to calculate the relative levels and p-values of miR-576-3p for SLE or RA patients as compared to controls. Unpaired two tailed t-test was used and error bars represent SD. *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Recombinant human IFNβ was obtained from R&D systems.

Techniques: Expressing, Two Tailed Test

Journal: Biochemical Journal

Article Title: Suppression of interferon β gene transcription by inhibitors of bromodomain and extra-terminal (BET) family members

doi: 10.1042/BJ20141523

Figure Lengend Snippet: RAW cells were incubated for 1 h without (−) or with (+) 1.0 μM JQ1 or 1.0 μM BI2536 and then stimulated for 8 h without (−) or with (+) poly(I:C) (10 μg/ml) ( A and C ) or for 4 h without (−) or with (+) LPS (100 ng/ml) ( B and D ). At each time point, the total RNA was extracted from the cells and Ifnb mRNA was quantified by qPCR ( A and B ) and the concentration of IFNβ in the cell culture medium was determined by ELISA ( C and D ). Results are means+S.E.M for triplicate determinations. ( A ) and ( B ) show the fold increase in mRNA levels relative to the values measured in cells that had not been stimulated with LPS or poly(I:C).

Article Snippet: The level of secreted IFNβ in the cell culture medium was determined using the Verikine mouse and human IFNβ ELISA kits (PBL Interferon Source) or the LEGEND MAXTM Mouse IFN-β ELISA Kit (BioLegend) following the manufacturer's protocol.

Techniques: Incubation, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Biochemical Journal

Article Title: Suppression of interferon β gene transcription by inhibitors of bromodomain and extra-terminal (BET) family members

doi: 10.1042/BJ20141523

Figure Lengend Snippet: ( A and B ) Gen2.2 cells were incubated for 1 h with or without BI-2536 (1.0 μM), JQ1 (1.0 μM) or I-BET151 (1.0 μM) and then stimulated for 8 h with CL097 (1.0 μg/ml) ( A ) or for 12 h with ODN1826 (1.0 μM) ( B ). The concentration of IFNβ in the culture medium was measured by ELISA. Results are means+S.D. from two independent experiments each performed in duplicate. ( C ) Gen2.2 cells were incubated for 1 h with or without BI-2536 (1.0 μM), JQ1 (1.0 μM), I-BET151 (1.0 μM) or BI-605906 (5.0 μM) and then stimulated for 1 h with CL097. Staining with anti-IRF5, or DAPI to reveal nuclei, followed by deconvolution microscopy was performed as described in the Materials and methods section. ( D ) Gen2.2 cells were incubated for 1 h with BI-2536 (1 μM) or JQ1 (1 μM), then stimulated for 1 h with the TLR7 agonist CL097 (1 μg/ml), cross-linked and lysed. Chromatin was sheared by sonication and ChIP was performed using anti-IRF5. The enrichment of the Ifnb promoter was measured by qPCR, normalizing to input. Results are means+S.D. similar results were obtained in three independent experiments each performed in duplicate. IP, immunoprecipitation.

Article Snippet: The level of secreted IFNβ in the cell culture medium was determined using the Verikine mouse and human IFNβ ELISA kits (PBL Interferon Source) or the LEGEND MAXTM Mouse IFN-β ELISA Kit (BioLegend) following the manufacturer's protocol.

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Microscopy, Sonication, Immunoprecipitation

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: ADAM17 Boosts Cholesterol Efflux and Downstream Effects of High-Density Lipoprotein on Inflammatory Pathways in Macrophages

doi: 10.1161/atvbaha.121.315145

Figure Lengend Snippet: Figure 1. Proinflammatory and anti-inflammatory responses in macrophages from APOA1Tg; Ldlr−/− mice mimic those of HDL (high-density lipoprotein). Peritoneal cells from male Ldlr−/− and APOA1Tg; Ldlr−/− control littermate mice were collected 4 days after thioglycolate injection. Macrophages were isolated from other cell types using a macrophage isolation kit and were further purified by a 1 h adhesion protocol before stimulation with LPS (lipopolysaccharide) or IFNβ (interferon β). A, Effect of LPS (10 ng/mL, 4 h) on inflammatory gene expression in macrophages isolated from Ldlr−/− and APOA1Tg; Ldlr−/− mice (n=5). B, Conditioned media were collected at indicated times from LPS-stimulated macrophages to analyze the release of TNF (tumor necrosis factor) α and CXCL1 (C-X-C motif chemokine ligand 1; n=4). C, Effect of LPS on the type 1 IFN-inducible genes Ifit2 and Mx1 analyzed in peritoneal macrophages from Ldlr−/− and APOA1Tg; Ldlr−/− mice (n=5–6). D, Effect of IFNβ on Ifit2 and Irf7 mRNA levels analyzed in peritoneal macrophages from Ldlr−/− and APOA1Tg; Ldlr−/− mice (n=5). E, Effect on STAT1 (signal transducer and activator of transcription 1) phosphorylation in IFNβ-stimulated peritoneal macrophages isolated from Ldlr−/− and APOA1Tg; Ldlr−/− mice (n=4). Phospho-STAT1 band intensity was normalized to that of total STAT1 and quantified (bar graph on the right). β-actin was used as an additional loading control. F, Representative photos and quantification (right) of lipid raft staining in thioglycolate-elicited peritoneal macrophages from Ldlr−/− and APOA1Tg; Ldlr−/− mice (n=7–13). G, BMDMs from female C57BL/6J mice were pretreated with HDL (100 μg/ mL) for 18 h. The cells were then stimulated with LPS (10 ng/mL) in the absence of HDL for 4 h, and inflammatory gene expression (Tnfa and Ifit2) was determined using quantitative polymerase chain reaction (n=4–7). H, BMDMs from male C57BL/6J mice were pretreated with HDL for 18 h. The cells were then stimulated with LPS (10 ng/mL) in the absence of HDL for 4 h to determine the phosphorylation status of p38 MAPK and STAT1 by immunoblot (n=4–5). Band intensities of p-p38 MAPK and p-STAT1 were normalized to those of total p38MAPK and STAT1, respectively, and quantified (bar graphs on the right). Data are shown as mean±SEM. Tests for normality (Shapiro-Wilk) and equal variance (Brown-Forsythe) were performed for each of the data sets. P values were determined accordingly by Kruskal-Wallis followed by Dunn multiple comparison tests (A), 1-way ANOVA followed by Tukey multiple comparison tests (C—Ifit2, D, E, H), Brown-Forsythe ANOVA followed by Dunnett multiple comparison tests (C—Mx1), 2-way ANOVA followed by Sidak multiple comparison test (B and G), or unpaired 2-tailed nonparametric Mann-Whitney test (F). Data are representative of at least 3 independent experiments performed in replicates. APOA1 indicates apolipoprotein A1.

Article Snippet: BMDMs were isolated and cultured as described previoulsy.37 After 7 days, BMDMs were treated with HDL isolated from APOA1Tg; Ldlr−/− mice in RPMI1640 medium containing 2% FBS for 18 hours, followed by washing of the cells and stimulation with ultrapure LPS, recombinant mouse IFNβ, TNFα (20 ng/mL; R&D systems; 410-MT), R848 (2 μg/mL, InvivoGen; tlrl-r848), or Poly:IC (10 μg/mL, Tocris Bioscience; 4287) for the indicated periods of time in the presence of 30% L929conditioned medium.

Techniques: Control, Injection, Isolation, Purification, Gene Expression, Phospho-proteomics, Staining, Real-time Polymerase Chain Reaction, Western Blot, Comparison, MANN-WHITNEY

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: Frontiers in Veterinary Science

Article Title: Interleukin-1β in tendon injury enhances reparative gene and protein expression in mesenchymal stem cells

doi: 10.3389/fvets.2022.963759

Figure Lengend Snippet: IL-1β and IL-6 are the predominant inflammatory cytokines present following surgically induced equine SDFT injury. Descriptive report of (A) inflammatory cytokines and (B) growth factors FGF-2 and TGF-β1,−2, and−3 in tendon ultrafiltrate were measured by ELISA following bilateral surgically induced SDFT injury of the forelimb over 21 days in 6 horses. Due to insufficient volume, samples from days 8 onward were grouped for TGF-β isoform analysis. When present, the dashed line at the y-axis represents the lower limit of quantification (LLoQ) for that cytokine or growth factor as determined by the assay manufacturer (MilliporeSigma, MA, USA). Concentrations measured below the LLoQ were assigned a value of 0.1 of the LLoQ.

Article Snippet: Protein expression of IL-1β licensed MSCs were evaluated with ELISA assays for equine IL-6 (R&D Systems, Minneapolis, MN, USA), human PGE2 (Enzo Biochem, Inc., Farmingdale, NY, USA), and equine VEGF (Kingfisher Biotech, Saint Paul, MN, USA) and performed per manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Veterinary Science

Article Title: Interleukin-1β in tendon injury enhances reparative gene and protein expression in mesenchymal stem cells

doi: 10.3389/fvets.2022.963759

Figure Lengend Snippet: IL-1β licensed equine MSCs produce significantly more IL-6, VEGF, and PGE2 than naïve MSCs. From RNA sequencing data, three proteins known to have tendon-relevant effects were selected to confirm downstream protein expression. Analysis of MSC cell culture supernatants by ELISA assay confirmed increased protein expression of IL-6, VEGF, and PGE2 following IL-1β licensing compared to naïve MSC controls. A one-tailed paired t -test was used to examine difference in protein expression between groups.

Article Snippet: Protein expression of IL-1β licensed MSCs were evaluated with ELISA assays for equine IL-6 (R&D Systems, Minneapolis, MN, USA), human PGE2 (Enzo Biochem, Inc., Farmingdale, NY, USA), and equine VEGF (Kingfisher Biotech, Saint Paul, MN, USA) and performed per manufacturer's instructions.

Techniques: RNA Sequencing, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, One-tailed Test