Journal: Breast Cancer (Tokyo, Japan)

Article Title: PIM1 is responsible for IL-6-induced breast cancer cell EMT and stemness via c-myc activation

doi: 10.1007/s12282-019-00966-3

Figure Lengend Snippet: IL-6 induces PIM1 expression in breast cancer cells. a T47D and MCF7 cells were exposed to 10 ng/ml IL-6 for 24 h. mRNA level of PIM1 was examined by qRT-PCR. b The protein level of PIM1 was examined by western bolt after IL-6 treatment. The mRNA ( c ) and protein ( d ) levels of PIM1 in T47D cells treated with IL-6 in gradient concentration were examined by qRT-PCR and western blot, respectively. The mRNA ( e ) and protein ( f ) levels of PIM1 in T47D cells treated with 10 ng/ml IL-6 for indicated time were examined by qRT-PCR and western blot, respectively

Article Snippet: IL-6 (HY-P7044), STAT3 inhibitor WP1066 (HY-15312) was obtained from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Concentration Assay

Journal: Breast Cancer (Tokyo, Japan)

Article Title: PIM1 is responsible for IL-6-induced breast cancer cell EMT and stemness via c-myc activation

doi: 10.1007/s12282-019-00966-3

Figure Lengend Snippet: IL-6 transcriptional activates PIM1 by stimulating STAT3. a PIM1 and STAT3 status were examined by western blot after treating with WP1066 at the presence or absence of IL-6 stimulation in T47D and MCF7 cells. b Transcriptional activity of PIM1 in response to IL-6 was measured by luciferase reporter assay with or without STAT3 activation inhibition

Article Snippet: IL-6 (HY-P7044), STAT3 inhibitor WP1066 (HY-15312) was obtained from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Western Blot, Activity Assay, Luciferase, Reporter Assay, Activation Assay, Inhibition

Journal: Breast Cancer (Tokyo, Japan)

Article Title: PIM1 is responsible for IL-6-induced breast cancer cell EMT and stemness via c-myc activation

doi: 10.1007/s12282-019-00966-3

Figure Lengend Snippet: PIM1 is essential for IL-6-induced breast cancer cell EMT and stemness. a PIM1 and EMT markers were examined by western blot in IL-6-treated T47D cells followed by PIM1 knocking down. b Cell invasion ability was examined in IL-6-treated T47D cells followed by PIM1 knocking down. c Indicated proteins were examined in IL-6-treated MCF7 cells followed by PIM1 knocking down. d Cell invasion ability of IL-6-treated MCF7 cells followed by PIM1 knocking down. The mRNA levels of EMT markers ( e ) and stemness markers ( f ) were measured by qRT-PCR in IL-6-treated T47D cells followed by PIM1 knocking down. E-cad, E-cadherin; Vim, vimentin; n-cad, N-cadherin

Article Snippet: IL-6 (HY-P7044), STAT3 inhibitor WP1066 (HY-15312) was obtained from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Western Blot, Quantitative RT-PCR

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: Clinical and Translational Medicine

Article Title: Recipient‐derived macrophages mediate acute cardiac allograft rejection via GSDMD‐induced pyroptosis mechanism

doi: 10.1002/ctm2.70729

Figure Lengend Snippet: Allografts transplanted into GSDMD −/− mice reduced pyroptosis and have prolonged survival. (A) PCR‐based genotyping of WT, GSDMD +/− , and GSDMD −/− mice. The WT allele was detected at 550 bp, and the knockout allele was detected at 423 bp. (B) Schematic illustration of BALB/c donor hearts transplanted into WT or Gsdmd−/− recipients on a C57BL/6 background. (C) Kaplan–Meier survival curves of cardiac grafts. n = 6 mice per group. (D) qPCR analysis of Il1b, Il18, Il6 and Tnfα mRNA expression in Day 5 grafts. n = 4 biologically independent samples per group. (E and F) Representative immunohistochemical staining and quantification of GSDMD, N‐GSDMD, IL‐1β and TNF‐α in Day 5 grafts. n = 4 biologically independent samples per group. (G–I) Representative flow cytometry plots and quantification of infiltrating CD45 + leukocytes in Day 5 grafts. n = 5 biologically independent samples per group. (J–L) Representative flow cytometry plots and quantification of infiltrating CD8 + T cells in Day 5 grafts. n = 5 biologically independent samples per group. (M–O) Representative flow cytometry plots and quantification of infiltrating macrophages in Day 5 grafts. n = 5 biologically independent samples per group. Data are presented as mean ± SEM. ns, not significant; * p < .05; ** p < .01; *** p < .001.

Article Snippet: RAW264.7 macrophages and HEK293T cells were cultured in DMEM supplemented with 10% foetal bovine serum and 1% penicillin‐streptomycin at 37°C in 5% CO. RAW264.7 cells were stimulated with recombinant mouse TNF‐α (MCE, HY‐P7090) at 20 or 40 ng/mL, or recombinant mouse IL‐6 (MCE, HY‐P7063) at 20 or 40 ng/mL, for 24 h. For subsequent mechanistic assays, TNF‐ and IL‐6 were used at 40 ng/mL each unless otherwise indicated.

Techniques: Knock-Out, Expressing, Immunohistochemical staining, Staining, Flow Cytometry

Journal: Clinical and Translational Medicine

Article Title: Recipient‐derived macrophages mediate acute cardiac allograft rejection via GSDMD‐induced pyroptosis mechanism

doi: 10.1002/ctm2.70729

Figure Lengend Snippet: GSDMD‐deficient macrophages attenuated CD8 + T cell recruitment and activation through IL‐1β. (A) Schematic illustration of the macrophage–CD8 + T cell Transwell co‐culture assay. BALB/c donor hearts were transplanted into GSDMD‐WT or GSDMD‐CKO recipients. On post‐operative Day 5, CD68 + macrophage‐enriched cells were isolated from cardiac grafts and spleens. CD8 + T cells were isolated from WT C57BL/6 spleens and activated with anti‐CD3/CD28 before co‐culture. Activated CD8 + T cells were seeded in the upper chamber, and macrophages with TNF‐α and IL‐6 stimulation were placed in the lower chamber. (B) Representative flow cytometry plots showing the percentage of CD8 + T cells among cells collected from the lower chamber after Transwell co‐culture. (C) Representative flow cytometry plots showing TNF‐α + CD8 + T cells among cells collected from the lower chamber after Transwell co‐culture. (D) Quantification of the number of migrated CD8 + T cells in the lower chamber. n = 6 biologically independent samples per group. (E) Quantification of TNF‐α + CD8 + T cells in the lower chamber. n = 6 biologically independent samples per group. Data are presented as mean ± SEM. *** p < .001; **** p < .0001.

Article Snippet: RAW264.7 macrophages and HEK293T cells were cultured in DMEM supplemented with 10% foetal bovine serum and 1% penicillin‐streptomycin at 37°C in 5% CO. RAW264.7 cells were stimulated with recombinant mouse TNF‐α (MCE, HY‐P7090) at 20 or 40 ng/mL, or recombinant mouse IL‐6 (MCE, HY‐P7063) at 20 or 40 ng/mL, for 24 h. For subsequent mechanistic assays, TNF‐ and IL‐6 were used at 40 ng/mL each unless otherwise indicated.

Techniques: Activation Assay, Co-culture Assay, Isolation, Co-Culture Assay, Flow Cytometry

Journal: Clinical and Translational Medicine

Article Title: Recipient‐derived macrophages mediate acute cardiac allograft rejection via GSDMD‐induced pyroptosis mechanism

doi: 10.1002/ctm2.70729

Figure Lengend Snippet: TNF‐α/IL‐6 induces GSDMD upregulation in macrophages via NF‐κB/STAT3 during acute rejection. (A) Volcano plot showing differentially expressed genes in macrophages between Day 1 and Day 5 after transplantation. (B) Pathway enrichment analysis showing activation of inflammatory pathways, including interferon responses, allograft rejection, TNF‐α signalling via NF‐κB, and IL‐6/JAK/STAT3 signalling. (C) Gene ontology analysis showing enrichment of biological processes related to type II interferon response, cellular response to cytokine stimulus, and positive regulation of cytokine production. (D and E) qPCR analysis of Stat3, Nfkb, Gsdmd, Il1b, Ifna and Ifng mRNA expression in RAW264.7 macrophages stimulated with TNF‐α or IL‐6. n = 3 independent experiments per group. (F) Western blot analysis of STAT3, p‐STAT3, p‐NF‐κB, GSDMD, N‐GSDMD, IFN‐α and IFN‐γ expression in RAW264.7 macrophages after TNF‐α or IL‐6 stimulation. (G and H) qPCR analysis of Gsdmd, Il1b, Ifna and Ifng mRNA expression in Day 5 allogeneic heart grafts after treatment with BAY 11–7082 or Stattic. n = 4 biologically independent samples per group. (I and J) Dual‐luciferase reporter assay showing STAT3‐induced GSDMD promoter activity and dose‐dependent activation of the GSDMD promoter by STAT3. n = 3 independent experiments per group. (K and L) Dual‐luciferase reporter assay showing NF‐κB‐induced GSDMD promoter activity and dose‐dependent activation of the GSDMD promoter by NF‐κB. n = 3 independent experiments per group. Data are presented as mean ± SEM. ** p < .01; *** p < .001; **** p < .0001.

Article Snippet: RAW264.7 macrophages and HEK293T cells were cultured in DMEM supplemented with 10% foetal bovine serum and 1% penicillin‐streptomycin at 37°C in 5% CO. RAW264.7 cells were stimulated with recombinant mouse TNF‐α (MCE, HY‐P7090) at 20 or 40 ng/mL, or recombinant mouse IL‐6 (MCE, HY‐P7063) at 20 or 40 ng/mL, for 24 h. For subsequent mechanistic assays, TNF‐ and IL‐6 were used at 40 ng/mL each unless otherwise indicated.

Techniques: Transplantation Assay, Activation Assay, Expressing, Western Blot, Luciferase, Reporter Assay, Activity Assay

Journal: Clinical and Translational Medicine

Article Title: Recipient‐derived macrophages mediate acute cardiac allograft rejection via GSDMD‐induced pyroptosis mechanism

doi: 10.1002/ctm2.70729

Figure Lengend Snippet: Proposed working model of macrophage GSDMD‐mediated pyroptosis in acute cardiac allograft rejection. After heart transplantation, recipient‐derived macrophages and CD8 + T cells progressively infiltrate the cardiac graft during the acute rejection stage. Recipient‐derived macrophages in acute rejection undergo M1‐like polarization under the influence of interferon signalling, IL‐6, and TNF‐α. These inflammatory signals activate NF‐κB/STAT3 signalling, leading to increased GSDMD and IL‐1β expression, GSDMD cleavage, N‐GSDMD pore formation, and IL‐1β release. NU6300 and disulfiram inhibit GSDMD‐mediated pyroptosis. Macrophage‐derived IL‐1β further enhances CD8 + T‐cell recruitment and activation, promoting immune crosstalk between macrophages and T cells and contributing to acute cardiac allograft rejection.

Article Snippet: RAW264.7 macrophages and HEK293T cells were cultured in DMEM supplemented with 10% foetal bovine serum and 1% penicillin‐streptomycin at 37°C in 5% CO. RAW264.7 cells were stimulated with recombinant mouse TNF‐α (MCE, HY‐P7090) at 20 or 40 ng/mL, or recombinant mouse IL‐6 (MCE, HY‐P7063) at 20 or 40 ng/mL, for 24 h. For subsequent mechanistic assays, TNF‐ and IL‐6 were used at 40 ng/mL each unless otherwise indicated.

Techniques: Transplantation Assay, Derivative Assay, Expressing, Activation Assay