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: 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: 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: Communications Biology

Article Title: IL-21 promotes the anti-tumor effect of anti-CD47 chimeric antigen receptor macrophages in ovarian cancer

doi: 10.1038/s42003-025-09331-x

Figure Lengend Snippet: A Schematic diagram of the CAR structure used in this study. The CAR mainly includes the CD47 scFv, the CD8α transmembrane and hinge region, and 4-1BB, with a CD3ζ intracellular signaling domain, with or without IL-21 linked by P2A. All CAR genes are linked to a GFP-expressing sequence. B , C IL-21 concentrations in CAR-M culture supernatants in the presence or absence of CD47 + ovarian cancer cell stimulation. D Phagocytosis assay results after flow cytometry-based CAR-M co-culture with SKOV3 cells (CD47 high and CD47 KO ) at a 1:1 ratio. E Phagocytosis assay results after flow cytometry-based CAR-M co-culture with A2780 cells (CD47 high and CD47 KO ) at a 1:1 ratio. F , G Flow cytometry-based results of tumor cell survival after the co-culture of CAR-Ms with SKOV3 or A2780 cells. All data were presented as the mean ± SEM of 3–4 experiments. Statistical analysis was performed using Student’s t -test or one-way ANOVA.

Article Snippet: The anti-CD47 CAR-M (IL-21) cell culture supernatant was collected, and the concentration of IL-21 (1112102, Dakewe Biotech) was determined according to the manufacturer’s instructions.

Techniques: Expressing, Sequencing, Cell Stimulation, Phagocytosis Assay, Flow Cytometry, Co-Culture Assay

Journal: Communications Biology

Article Title: IL-21 promotes the anti-tumor effect of anti-CD47 chimeric antigen receptor macrophages in ovarian cancer

doi: 10.1038/s42003-025-09331-x

Figure Lengend Snippet: A , B Expression of CD86 and CD206 in CAR-Ms after 48 h of co-culture with SKOV3 or A2780 cells, based on flow cytometry. C Activation of CTLs after CAR-M and CD3 + T cell co-culture with SKOV3 or A2780 cells, based on flow cytometry. D – I Detection of cytokines, including GzmB, PRF1, LT-α, TNF-α, IL-2, and IFN-γ, in the supernatants of CAR-M and CD3 + T cells co-cultured with SKOV3 or A2780 cells. J Flow cytometry results of FasL expression on the surfaces of CD3 + T cells after co-culture with CAR-Ms, SKOV3 cells, or A2780 cells. K Flow cytometry results of CD40L expression on the surfaces of CD3 + T cells after co-culture with CAR-Ms, SKOV3 cells, or A2780 cells. L IL-21 enhances CAR-M anti-tumor effects by promoting phagocytosis and CTL activation. All data are presented as the mean ± SEM of 3–4 experiments. Statistical analysis was performed using one-way ANOVA.

Article Snippet: The anti-CD47 CAR-M (IL-21) cell culture supernatant was collected, and the concentration of IL-21 (1112102, Dakewe Biotech) was determined according to the manufacturer’s instructions.

Techniques: Expressing, Co-Culture Assay, Flow Cytometry, Activation Assay, Cell Culture

Journal: Communications Biology

Article Title: IL-21 promotes the anti-tumor effect of anti-CD47 chimeric antigen receptor macrophages in ovarian cancer

doi: 10.1038/s42003-025-09331-x

Figure Lengend Snippet: A UMAP plot showing the major cell types. Dots represent individual cells, and colors represent different cell populations. B UMAP of each group; among them, CD47 CAR-Ms are orange, and CD47 CAR-M(IL-21) cells are blue. C Relative abundance of each cell type, as defined in A, as a percentage of the total single-cell transcriptomes captured. D Cluster-specific marker gene point map of CD47 CAR-M group. E Cluster-specific marker gene point map of CD47 CAR-M(IL-21) group. F Relative proportions of T cells, NK cells, CD8 + T cells, CD4 + T cells, and Treg cells between the two groups.

Article Snippet: The anti-CD47 CAR-M (IL-21) cell culture supernatant was collected, and the concentration of IL-21 (1112102, Dakewe Biotech) was determined according to the manufacturer’s instructions.

Techniques: Marker

Journal: Communications Biology

Article Title: IL-21 promotes the anti-tumor effect of anti-CD47 chimeric antigen receptor macrophages in ovarian cancer

doi: 10.1038/s42003-025-09331-x

Figure Lengend Snippet: A RNA-seq results of anti-CD47 chimeric antigen receptor-macrophages (CAR-Ms) and anti-CD47 CAR-M(IL-21) cells. B GO analysis of RNA-seq data from anti-CD47 CAR-Ms and anti-CD47 CAR-M(IL-21) cells. C Western blot (WB) results of ERK and AKT in THP-1Ms (THP-1 macrophages) after IL-21 stimulation. The data were representative of three independent experiments. D WB results of STAT1 and STAT3 in Jurkat cells after IL-21 stimulation. Data were representative of three independent experiments. E RNA-seq results based on SKOV3 cells after IL-21 stimulation. F GO analysis of RNA-seq data of IL-21-stimulated SKOV3 cells. All data were presented as the mean ± SEM of triplicate experiments. Two-tailed unpaired Student’s t -test was used.

Article Snippet: The anti-CD47 CAR-M (IL-21) cell culture supernatant was collected, and the concentration of IL-21 (1112102, Dakewe Biotech) was determined according to the manufacturer’s instructions.

Techniques: RNA Sequencing, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Airway immune profiles and therapeutic implications of IGF1 in eosinophilic granulomatosis with polyangiitis

doi: 10.1038/s41467-025-68104-6

Figure Lengend Snippet: a Immunofluorescent staining and quantification of IGF1⁺ epithelial cells at bronchial mucosae from control ( n = 6), SEA ( n = 9), EGPA ( n = 10), Cs-remission ( n = 3), and Cs-relapse ( n = 5) groups (biological replicates). Scale bar: 100 μm. b Bar plots of IGF1 concentrations in sputum samples from Control ( n = 13), SEA ( n = 23), and EGPA ( n = 21) (biological replicates). c Schematic of EGPA airway epithelial cells in ALI culture stimulated with IL-13, IL-33, or medium (Control). Bar plots show relative expression of IGF1 , IGF1R , and IGFBP3 , and IGF1 concentrations under different conditions. Data from 3 independent experiments (biological replicates). d Schematic of ALI system with IGF1 stimulation versus control. Representative histological (HE, PAS) and immunofluorescent (MUC5AC with DAPI) staining demonstrate morphological changes and mucin production (goblet hyperplasia) in ALI cultures with medium (Control) or IGF1 stimulation. Scale bars: 50 μm (HE, PAS), 20 μm (MUC5AC). Bar plots of IL-25, IL-33, and TSLP concentrations in culture supernatant at time points (Day 9, 13, 17, 21). Data from 3 independent experiments (biological replicates). e Scatter plot showing positive correlation between eosinophil abundance and IGF1 concentration in sputum from EGPA patients ( n = 21). f Schematic of proposed mechanism where IGF1 promotes goblet hyperplasia and augments T2-mediated inflammation through IGF1-IL25 loop, contributing to disease exacerbation in asthma and EGPA. Data presented as mean ± SD. a – c Two-sided one-way ANOVA with Tukey’s post-hoc test. d Two-sided unpaired t -test. e Two-sided Pearson correlation test. * P < 0.05, ** P < 0.01, *** P < 0.001; NS, not significant. Exact P values in Supplementary Data . ALI air-liquid interface, HE hematoxylin and eosin, IGF1R insulin-like growth factor 1 receptor, IGFBP3 insulin-like growth factor binding protein 3, PAS periodic acid-Schiff, TSLP thymic stromal lymphopoietin.

Article Snippet: Mice were administered 50 μg of low-endotoxin HDM (Greer Labs, Cat# XPB91D3A2.5) combined with 500 ng recombinant mouse IL-33 (PEPROTECH, Cat# 210-33) via intranasal injection (i.n.) at Day 0, Day 7, Day 14 and Day 21, and recombinant IL−25 (2 μg in 50 μL saline, recombinant mouse IL25, R&D System, Cat# 1399-IL-025) was administered via intranasal injection (i.n.) at Day 15, Day 17, Day 19, and Day 21, Mice were sacrificed at Day 24, and tissues were harvested for downstream analysis.

Techniques: Staining, Control, Expressing, Concentration Assay, Binding Assay

Journal: Nature Communications

Article Title: Airway immune profiles and therapeutic implications of IGF1 in eosinophilic granulomatosis with polyangiitis

doi: 10.1038/s41467-025-68104-6

Figure Lengend Snippet: a – e Anti-IGF1 treatment reduces eosinophilic inflammation and airway remodeling in IL-5 transgenic mice challenged with HDM and IL-33. Mice were divided into Control (PBS-treated), Model (HDM+IL-33 challenged), and Anti-IGF1 (HDM+IL-33+anti-IGF1 antibody) groups. a Total cell counts in bronchoalveolar lavage fluid (BALF) from control, model, and anti-IGF1-treated groups. b Eosinophil percentage in BALF. c Absolute eosinophil counts in BALF. d IL−25 levels in BALF quantified by ELISA. e Representative histological images of lung sections stained with hematoxylin and eosin (H&E, upper panels) and periodic acid-Schiff (PAS, lower panels), with corresponding inflammation and PAS score quantifications. Scale bar, 100 μm. f – h IGF1R deficiency modulates eosinophilic inflammation and airway remodeling via IL−25 in HDM+IL-33 challenged mice. f Total cell counts in BALF from wild-type (WT, Scgb1a1 -IRES-+/+ Igf1r f/f ), conditional knockout ( Scgb1a1 -IRES-Cre/+ Igf1r f/f , CKO), and CKO mice treated with recombinant IL-25 (rIL-25). All groups were challenged with HDM+IL-33. g Flow cytometric analysis of eosinophils (CD45 + Siglec-F + CD11c - ) in BALF. Representative plots and quantification of eosinophil percentages are shown. h Representative H&E (upper panels) and PAS (lower panels) staining of lung sections from WT, CKO, and CKO+rIL-25 groups, with quantification of inflammation and PAS scores. Scale bar, 100 μm. Statistical analysis: Data are presented as mean ± SD ( n = 5 mice per group). Statistical significance was determined using a two-sided one-way ANOVA with Tukey’s post hoc test. * p < 0.05; ** p < 0.01; *** p < 0.001; NS, not significant. Exact P values and complete test statistics for a – h are provided in Supplementary Data . CKO conditional knockout, HDM house dust mite, rIL-25 recombinant IL-25, WT wild-type.

Article Snippet: Mice were administered 50 μg of low-endotoxin HDM (Greer Labs, Cat# XPB91D3A2.5) combined with 500 ng recombinant mouse IL-33 (PEPROTECH, Cat# 210-33) via intranasal injection (i.n.) at Day 0, Day 7, Day 14 and Day 21, and recombinant IL−25 (2 μg in 50 μL saline, recombinant mouse IL25, R&D System, Cat# 1399-IL-025) was administered via intranasal injection (i.n.) at Day 15, Day 17, Day 19, and Day 21, Mice were sacrificed at Day 24, and tissues were harvested for downstream analysis.

Techniques: Transgenic Assay, Control, Enzyme-linked Immunosorbent Assay, Staining, Knock-Out, Recombinant

Journal: eLife

Article Title: Transcriptional correlates of malaria in RTS,S/AS01-vaccinated African children: a matched case–control study

doi: 10.7554/eLife.70393

Figure Lengend Snippet:

Article Snippet: Antibody , anti-IL-21, clone 3A3-N2 (mouse monoclonal) , Miltenyi Biotec , Cat# 130-120-702 , Fluorochrome: APC.

Techniques: Staining, Concentration Assay, Recombinant, Produced, Sequencing, Isolation, Gel Extraction, Software, Adhesive, Reverse Transcription, Magnetic Beads