Journal: International journal of antimicrobial agents

Article Title: Antibacterial and antivirulence activities of auranofin against Clostridium difficile

doi: 10.1016/j.ijantimicag.2018.09.018

Figure Lengend Snippet: The minimum inhibitory concentration (MIC, µg/mL) of auranofin and control drugs against vancomycin-resistant Enterococcus faecium isolates.

Article Snippet: Auranofin, linezolid (Chem-Impex International, Wood Dale, IL), vancomycin hydrochloride (Gold Biotechnology, St. Louis, MO) metronidazole (Beantown Chemical Corporation, Hudson, NH), sodium selenite (MP Biomedicals, Santa Ana, CA), and fidaxomicin (Apexbio, Houston, TX) were procured from commercial vendors.

Techniques: Concentration Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Tadalafil Reduces Myeloid-Derived Suppressor Cells and Regulatory T Cells and Promotes Tumor Immunity in Patients with Head and Neck Squamous Cell Carcinoma

doi: 10.1158/1078-0432.CCR-14-1711

Figure Lengend Snippet: MDSCs identification. A, the indicated myeloid cell subsets were tested for suppressive activity against CFSE-labeled autologous T cells stimulated with beads coated with anti-CD3/anti-CD28 antibodies. Data normalized on the control (no MDSC) are cumulative of five independent experiments using PBMCs from 5 patients. P value for the ANOVA test (Pa) and the Tukey post hoc test are reported. B, example of multicolor FACS analysis for MDSC phenotype CD33+IL4Rα+ cells are highlighted in blue. C, intratumoral CD33+ IL4Rα+ cells were retrospectively evaluated in the tumor specimen of recurrent or nonrecurrent OSCC patients by immunofluorescence microscopy. P value for t test is reported.

Article Snippet: FACS sorting For the suppressive assay, cryoconserved PBMCs were thawed and stained with Percp-Cy5.5–conjugated anti-human HLADR, FITC-conjugated anti-human CD33 (BD) and PE-conjugated anti-human IL4Rα (R&D Systems).

Techniques: Activity Assay, Labeling, Control, Immunofluorescence, Microscopy

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Tadalafil Reduces Myeloid-Derived Suppressor Cells and Regulatory T Cells and Promotes Tumor Immunity in Patients with Head and Neck Squamous Cell Carcinoma

doi: 10.1158/1078-0432.CCR-14-1711

Figure Lengend Snippet: An intermediate tadalafil dose modulates most effectively tumor immunity. The ratio between the MDSCs (A) or the log2-ratio of the CD8 proliferation (B) after (t2) and before (t1) pharmacologic treatment was plotted against the weight-normalized tadalafil dose. Best-fitting quadratic curve and confidence interval (gray area) are reported. cGMP (C) and cAMP (D) were measured by ELISA in the following FACS-sorted cell population from patients (n = 3) treated with intermediate or high dosage of tadalafil: CD33+IL4Rα+ (MDSCs), HLADRhigh (APC), or CD3+ (T cells). Pt, paired t test; BDL, below detection limit.

Article Snippet: FACS sorting For the suppressive assay, cryoconserved PBMCs were thawed and stained with Percp-Cy5.5–conjugated anti-human HLADR, FITC-conjugated anti-human CD33 (BD) and PE-conjugated anti-human IL4Rα (R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Tadalafil Reduces Myeloid-Derived Suppressor Cells and Regulatory T Cells and Promotes Tumor Immunity in Patients with Head and Neck Squamous Cell Carcinoma

doi: 10.1158/1078-0432.CCR-14-1711

Figure Lengend Snippet: Tadalafil modulates tumor microenvironment. CD33/IL4Rα (A), CD4/FoxP3 (B), or CD8/CD69 (C) intratumoral concentration was evaluated by immune-fluorescence microscopy. Pa, P ANOVA test.

Article Snippet: FACS sorting For the suppressive assay, cryoconserved PBMCs were thawed and stained with Percp-Cy5.5–conjugated anti-human HLADR, FITC-conjugated anti-human CD33 (BD) and PE-conjugated anti-human IL4Rα (R&D Systems).

Techniques: Concentration Assay, Fluorescence, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: Tg ROP18 I interacted with TRIM21’s PRY-SPRY domain. (A) The Co-IP result indicated the interaction of Tg ROP18 I and TRIM21. (B,C) The Co-IP result indicated that ROP18-KD still bound with TRIM21. (D) Sketch map of TRIM21 WT and truncation mutants. (E) The Co-IP results showed the PRY-SPRY domain of TRIM21 was indispensable for Tg ROP18 I -TRIM21 interaction. The experiments were repeated three times. The values were analyzed using the one-way ANOVA. Data were expressed as the mean ± SEM (** p < 0.01; *** p < 0.001; IP, immunoprecipitation).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: Tg ROP18 I promoted the phosphorylation of TRIM21. (A,B) HEK293T cells were transfected with different amount of pcDNA3.1-ROP18 I -FLAG as indicated, and the cell lysates were subjected to TRIM21 IP and Western blotting. The more plasmids were transfected to HEK293T cells, the higher levels of phosphorylated TRIM21 were observed, and the differences between groups were significant. (C,D) HFF cells were infected with CEP or CEP- rop18 I , and then the total protein was extracted and subjected to TRIM21 IP and Western blotting. The results showed that more phosphorylated TRIM21 was detected in the CEP- rop18 I infected cells than which detected in the CEP infected cells and uninfected cells, while the phosphorylation levels of TRIM21 were not significantly different between the CEP infected and uninfected cells. The experiments were repeated three times. The values were analyzed using the one-way ANOVA. Data were expressed as the mean ± SEM (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

Techniques: Phospho-proteomics, Transfection, Western Blot, Infection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: Tg ROP18 I promoted TRIM21 degradation through lysosomal pathway. (A) HEK293T cells were transfected with the increased amounts of pcDNA3.1-ROP18 I -FLAG as indicated. (B) HEK293T cells were co-transfected with a stable amount of pcDNA3.1-TRIM21-HA and the increased amounts of pcDNA3.1-ROP18 I -FLAG as indicated. The endogenous or overexpressed TRIM21 level was decreased with the increased ROP18 level. (C) HEK293T cells were co-transfected with pcDNA3.1-TRIM21-HA and pcDNA3.1-ROP18 I -FLAG or pcDNA3.1-ROP18 I -KD-FLAG as indicated. The results of Western blotting detection with the cell lysates indicated that much more TRIM21 was detected in the ROP18-KD overexpression group than in the ROP18 overexpression group. (D) Lysates of HFFs infected with RH or RH-△ rop18 was detected by Western blotting, and more TRIM21 was detected in the RH-△ rop18 infection group than in the RH infection group. (E) HEK293T cells were co-transfected with 1mg of pcDNA3.1-TRIM21-HA and increased amounts of pcDNA3.1-ROP18 I -FLAG. The cells were treated with MG132 or Leupeptin, or left untreated. Cell lysates were subjected to Western blotting, and the results showed that TRIM21’s level was decreased with the increased amount of Tg ROP18 I in the cells treated with MG132 or DMSO. However, TRIM21’s level was kept stable in the Leupeptin treated group. All the experiments were repeated three times. IB, immunoblot.

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

Techniques: Transfection, Western Blot, Over Expression, Infection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: TRIM21 transcription and translation levels were upregulated following T. gondii infection. HFF cells infected with RH or CEP were harvested for detection of TRIM21 transcription and translation level. (A) Comparison of the TRIM21 transcription levels between the indicated groups with qRT-PCR. The CEP infection resulted in the highest TRIM21 transcription level, followed by RH infection, and uninfection at 1 h post infection, the differences between groups were significant. At 24 h post infection, the RH infection resulted in a significant higher TRIM21 transcription level than in CEP infection or uninfection groups between which no significant difference was observed in TRIM21 transcription level. (B,C) Comparison of the translation level of TRIM21 in the HFF cells infected with RH or CEP for the indicated time with Western blot (up panels). The densitometrical analysis for the intensity of TRIM21 bands normalized to its corresponding GAPDH intensity showed that, both CEP and RH infection resulted in significant higher transcription levels of TRIM21 than in uninfected cells (down panels). All the experiments were repeated four times. The values were analyzed using the one-way ANOVA. Data were expressed as the mean ± SEM (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

Techniques: Infection, Comparison, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: TRIM21 is involved in the IFN-γ induced inhibition of CEP proliferation (but not RH proliferation) in HFFs. The proliferation of RH and CEP tachyzoies in the HFFs was evaluated after stimulation with IFN-γ for 24 h (A–C) , or transfection with pcDNA3.1-TRIM21-HA for 24 h (E–F) ; and proliferation of CEP-WT was evaluated after transfection with si-TRIM21 for 48 h in HFFs followed by IFN-γ treatment for 24 h (H–I) . TRIM21 protein levels were measured by Western blotting (A,D,G) . The average number of tachyzoites in 100 parasitophorous vacuoles (PVs) was counted (B,E,H) , and the percentage of the PVs containing 1, 2, 4, or 8 parasites was determined by immune fluorescence assay (C,F,I) . (A) The TRIM21 protein level was significantly up-regulated under IFN-γ stimulation in a dose-dependent manner, with the response concentration ranged from 50 to 500 U/ml. (B,C) The proliferation of the RH and CEP tachyzoites in HFFs was significantly inhibited after IFN-γ stimulation. (D) The overexpression of TRIM21 was detected. (E,F) The overexpression of TRIM21 significantly inhibited the CEP proliferation, but not affected RH proliferation. (G) The si-TRIM21 transfection significantly inhibited the TRIM21 translation. (H,I) The IFN-γ induced inhibition of CEP proliferation was relieved by TRIM21 knockdown. All the experiments were repeated three times. The values were analyzed using the one-way ANOVA and two-way ANOVA. Data were expressed as the mean ± SEM (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

Techniques: Inhibition, Transfection, Western Blot, Fluorescence, Concentration Assay, Over Expression, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: TRIM21 knockdown relieves the IFN-γ-induced ubiquitin labeling on CEP parasitophorous vacuole membrane (PVM) in HFFs. (A,B) HFFs were transfected with negative control siRNA (si-NC) or siRNA specific against TRIM21 (si-TRIM21), and stimulated with IFN-γ or not as indicated. After T. gondii infection, HFFs were subjected to immunofluorescence assay (IFA). IFN-γ induced ubiquitin labeling on the CEP PVM, but this labeling was relieved by TRIM21 knockdown regardless of IFN-γ simulation or not. (C,D) HFFs were stimulated with IFN-γ or not, and infected with CEP. The cells were then treated with LysoTracker ® (acidic dye) and subjected to immunofluorescence assay (IFA). The result showed us that IFN-γ induced acidification of CEP. On the left, a representative fluorescent image is shown for the T. gondii CEP strain expressing GFP. The yellow box inside each representative image is shown as magnified pictures nearby (A,C) . The percentage of vacuoles stained red with ubiquitin labeling or LysoTracker ® was shown in the right bar diagram (B,D) . Scale bar is 10 μm. The experiments were repeated three times. The values were analyzed using the one-way ANOVA or two-tailed unpaired Student t test. Data were expressed as the mean ± SEM (** p < 0.01; *** p < 0.001).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

Techniques: Knockdown, Ubiquitin Proteomics, Labeling, Membrane, Transfection, Negative Control, Infection, Immunofluorescence, Expressing, Staining, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: Tg ROP18 I relieved TRIM21 mediated inhibition of T. gondii proliferation regardless of strain types. (A–D) HFFs were transfected with pcDNA3.1-TRIM21-HA or pcDNA3.1(+) for control, and infected with RH-△ rop18 (A,B) or CEP- rop18 I (C,D) parasites as indicated. Parasitic proliferation was measured at 18 h (RH-△ rop18 ) or 24 h (CEP- rop18 I ) post-infection. The average number of tachyzoites in 100 vacuoles (A,C) or the number of vacuoles containing 1, 2, 4, or 8 parasites (B,D) was determined by fluorescence microscopy. The results indicated that TRIM21 overexpression inhibited the RH-△ rop18 multiplication, but had no significant effect on CEP- rop18 I multiplication. The experiments were repeated three times. The values were analyzed using the two-tailed unpaired Student t test and two-way ANOVA. Data were expressed as the mean ± SEM (** p < 0.01).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

Techniques: Inhibition, Transfection, Control, Infection, Fluorescence, Microscopy, Over Expression, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: TRIM21 mediated NF-κB activation. (A,B) In the HFF cells stimulated with the indicated concentrations of IFN-γ for 24 h, the p-p65 (S536) level was significantly higher than that in the untreated group. (C,D) Western-blot detection of p-p65 (S536) level showed that TRIM21 overexpression significantly elevated p-p65 (S536) phosphorylation in dose-dependent manner. (E,F) In the siRNA-TRIM21 transfected groups, TRIM21 expression was suppressed, but no significant difference was found in the p-p65 (S536) levels between the TRIM21 knockdown group with or without IFN-γ induction. The experiments were repeated three times. The values were analyzed using the one-way ANOVA and the data were expressed as the mean ± SEM (** p < 0.01; *** p < 0.001).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

Techniques: Activation Assay, Western Blot, Over Expression, Phospho-proteomics, Transfection, Expressing, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: The interaction of p65 and IκB-α was suppressed with TRIM21 overexpression. Interaction of TRIM21 and IκB-α in the total cell lysates of HEK293T cells were analyzed by IP and Western-blot after either treatment with IFN-γ or transfection with pcDNA3.1-TRIM21-HA for the indicated time. (A–C) The IP with the anti-TRIM21 antibody identified the interaction of IκB-α with the overexpressed and the endogenous TRIM21. TRIM21 overexpression promoted TRIM21-IκB-α interaction (A) . IFN-γ induction elevated TRIM21 production and promoted TRIM21-IκB-α interaction (B) . The TRIM21 production induced by IFN-γ increased with the prolonged treating time and promoted TRIM21-IκB-α interaction (C) . (D,E) The IP with the anti-p65 antibody identified the complex of IκB-α-TRIM21-p65 (D) , and the interaction of p65-ubiquitin (E) . The experiments were repeated three times. The TRIM21 band is indicated with “*”, and the IκB-α band is indicated with a black arrow.

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

Techniques: Over Expression, Western Blot, Transfection, Ubiquitin Proteomics

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: Tg ROP18 I targets host TRIM21 for immune escape. 1. Host IFN-γ-induced factor TRIM21 restricted T. gondii replication through NF-κB activation and TRIM21 overexpression suppressed the p65-IκB-α interaction to activate NF-κB pathway. 2. Tg ROP18 I which was discharged by T. gondii , interacted with the PRY-SPRY domain of human TRIM21, promoted TRIM21 phosphorylation, and induced TRIM21 degradation via lysosomal pathway. 3. IFN-γ induced ubiquitin labeling on the CEP PVM which resulted in PV acidification and death of parasites, but this labeling was relieved by TRIM21 knockdown regardless of IFN-γ simulation or not.

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

Techniques: Activation Assay, Over Expression, Phospho-proteomics, Ubiquitin Proteomics, Labeling, Knockdown

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: Nature Communications

Article Title: Stromal heterogeneity may explain increased incidence of metaplastic breast cancer in women of African descent

doi: 10.1038/s41467-023-41473-6

Figure Lengend Snippet: a Gating of tomato- (Tom - ) and tomato+ (Tom + ) populations of KTB34, KTB39, and co-cultured KTB40/KTB42 and KTB34/KTB39 cell lines ( n = 3). PZP cell lines are Tom + . b CD49f and EpCAM staining patterns of KTB34, KTB39, and co-cultured KTB40/KTB42 and KTB34/KTB39 cell lines ( n = 3). Isotype controls are shown in Fig. . c Quantification of CD49f + /EpCAM high , (KTB40 + 34* p = 0.002; KTB42 + 34* p = 0.0019; KTB40 + 39* p < 0.0001; KTB42 + 39* p < 0.0001), CD49f + /EpCAM med (KTB40 + 34* p = 0.0186; KTB42 + 34* p = 0.0124; KTB40 + 39* p = 0.0183), and CD49f + /EpCAM low (KTB40 + 34* p = 0.0006; KTB42 + 34* p = 0.0012; KTB40 + 39* p = 0.0185; KTB42 + 39* p = 0.0414) populations in Tom− cell population ( n = 3). d CD49f and EpCAM staining patterns of KTB40, KTB42, and co-cultured KTB40/KTB42 and KTB34/KTB39 cell lines. Only Tom + population was analyzed ( n = 3). e Quantification of CD49f + /EpCAM + population among Tom + population ( n = 3). (KTB40 + 34* p < 0.0001; KTB40 + 39* p = 0.0015; KTB42 + 34* p = 0.0002; KTB42 + 39* p = 0.0024) ( f ) pSTAT3 Y705, pSTAT3 S727, and STAT3 expression levels in KTB34 cells treated with CM obtained from KTB34, KTB40, and co-cultured KTB34 and KTB40 cells followed by IgG control and IL-6R neutralizing antibody treatment ( n = 3). CM was treated with IgG or IL-6R neutralizing antibodies for 1 h and CM treatment lasted for 2 h. β–actin was used as an internal control ( n = 3). In this representative experiment, the same batch of extracts was used to run four blots and each blot was probed with indicated antibodies separately. Statistical significance derived (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001) using Two-tailed t -test. All the data points are shown as mean ± SEM. Source data are provided as a .

Article Snippet: For IL-6 signaling neutralization studies, anti-IL-6R antibody (MAB227, R&D Systems) or control IgG (sc-2025, Santa Cruz Biotechnology) at 2 μg/ml concentration was added to CM for 1 h prior to adding CM to epithelial cells.

Techniques: Cell Culture, Staining, Expressing, Control, Derivative Assay, Two Tailed Test