Journal: The Journal of Clinical Investigation

Article Title: CTRP4/interleukin-6 receptor signaling ameliorates autoimmune encephalomyelitis by suppressing Th17 cell differentiation

doi: 10.1172/JCI168384

Figure Lengend Snippet: ( A ) Purified CD4 + T cells from Ctrp4 –/– and WT mice were stimulated with IL-6 (100 ng/mL) for indicated times. Lysates were subjected to Western blot analysis for indicated antibodies. ( B ) Freshly isolated WT and Ctrp4 –/– CD4 + T cells were treated with IL-6 (100 ng/mL) for 30 minutes. Levels of p-STAT3 were determined by flow cytometry. ( C ) Representative flow cytometry analysis of p-STAT3 and p-STAT1 of CD4 + T cells from Ctrp4 –/– and WT EAE-induced mice at the peak of disease. ( D – F ) WT CD4 + T cells were polarized to Th17. The intracellular IL-17A was analyzed by flow cytometry ( D ), and gene expression levels of Rorc mRNA were analyzed by quantitative real-time PCR ( E ). Supernatants were collected to determine levels of IL-17A by ELISA ( F ). ( G ) Purified WT naive CD4 + T cells were polarized under Th17 conditions with indicated doses of rhCTRP4. Quantification of the percentages of CD4 + IL17A + cells was analyzed by flow cytometry. ( H ) Purified WT CD4 + cells were activated with IL-6 (10 ng/mL) or rhCTRP4 (100 ng/mL) alone or treated with IL-6 prior to treatment with rhCTRP4 for 1 hour or without treatment as control. Western blot was performed to analyze the activation of STAT3. ( I ) Purified WT CD4 + cells were activated with IL-6 or with IL-6 pretreated with various concentrations of rhCTRP4 (100 ng/mL, 500ng/mL, and 1,000 ng/mL) followed by Western blot to analyze the activation of STAT3 and JAK2. The samples derived from the same experiment, and gels/blots were processed in parallel. Representative of 3 independent experiments with 5 mice per experiment. ( J ) Purified WT CD4 + cells were activated with IL-6 (10 ng/mL) prior to treatment with rhCTRP4 (100 ng/mL) for the indicated times. Time-dependent changes in the levels of p-STAT3 were evaluated by Western blot. Data are represented as mean ± SEM and are from 1 of 3 independent experiments with similar results. ( D – F ) Statistical significance was determined using paired Student’s t test; ( G ) statistical significance was determined using 1-way ANOVA with Tukey’s post test. * P < 0.05; ** P < 0.01.

Article Snippet: For pharmacological inhibition of STAT3, STAT3 inhibitor S3I-201 (Selleck, catalog S1155) was injected intraperitoneally at 5 mg/kg every 2 days.

Techniques: Purification, Western Blot, Isolation, Flow Cytometry, Gene Expression, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control, Activation Assay, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: CTRP4/interleukin-6 receptor signaling ameliorates autoimmune encephalomyelitis by suppressing Th17 cell differentiation

doi: 10.1172/JCI168384

Figure Lengend Snippet: ( A and B ) Proliferative response of Ba/F3–gp130–IL-6R cells cultured for 2 days in the presence of exogenous rhCTRP4 (100 ng/mL), OSM (100 ng/mL), IL-6 (10 ng/mL), IL-3 (10 ng/mL), rhCTRP4 plus IL-6, or OSM plus rhCTRP4, or left untreated ( A ). ( B ) Proliferative response of Ba/F3-gp130 cells cultured for 2 days with OSM (100 ng/mL), IL-6 (10 ng/mL), rhCTRP4 (100 ng/mL), hyper–IL-6 (10 ng/mL), a combination of IL-6 (10 ng/mL) and IL-6R (10 ng/mL), a combination of IL-6 and IL-6R plus rhCTRP4, hyper–IL-6 plus rhCTRP4, or IL-3 (10 ng/mL). The proliferation in indicated culture conditions was determined by the colorimetric CCK8 assay. Results are shown as RLUs and normalized to the growth of cells cultured in medium. ( C ) Purified WT CD4 + cells were activated with hyper–IL-6 (10 ng/mL) prior to treatment with rhCTRP4 for 1 hour or without treatment as control. Western blot was performed to analyze the activation of STAT3. ( D ) Naive CD4 + T cells were differentiated toward Th17 cells with TGF-β plus the combination of IL-6 (10 ng/mL) and IL-6R (10 ng/mL) in the presence or absence of rhCTRP4 (100 ng/mL) or differentiated toward Th17 cells with TGF-β and hyper–IL-6 (10 ng/mL) in the presence or absence of rhCTRP4 (100 ng/mL). Data are represented as mean ± SEM and are from 1 of 3 independent experiments with similar results. ( A , B , and D ) Statistical significance was determined using 1-way ANOVA with Tukey’s post test.** P < 0.01.

Article Snippet: For pharmacological inhibition of STAT3, STAT3 inhibitor S3I-201 (Selleck, catalog S1155) was injected intraperitoneally at 5 mg/kg every 2 days.

Techniques: Cell Culture, CCK-8 Assay, Purification, Control, Western Blot, Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: CTRP4/interleukin-6 receptor signaling ameliorates autoimmune encephalomyelitis by suppressing Th17 cell differentiation

doi: 10.1172/JCI168384

Figure Lengend Snippet: ( A ) After EAE induction of WT mice, 500 ng/mice rhCTRP4 or control (BSA) were administered intraperitoneally every day starting at disease onset on day 9 after immunization until sacrificed. The clinical scores were monitored daily and are depicted ( n = 10/group). ( B ) Representative images of H&E and LFB staining on spinal cord sections of mice at the peak of EAE. Scale bars: 200 μm. ( C ) CNS monocytes were harvested on day 18. The absolute cell numbers of indicated CNS-infiltrating cell populations (gated on CD45 + ) or the absolute numbers of CD4 + IFN-γ + , CD4 + IL-17A + , and CD4 + IL-17A + IFN-γ + in CNS were analyzed. ( D ) Representative immunofluorescent images and quantification of IL-17A + cells in the spinal cord of indicated mice at day 18 after immunization. Nuclei were counterstained with DAPI. Scale bars: 100 μm. ( E ) Representative immunohistochemistry detection and quantification of p-STAT3(Y705) in the spinal cords of indicated mice at day 18 after immunization. Scale bars: 100 μm. ( F ) CD4 + T cells were isolated from Ctrp4 –/– and WT mice 18 days after EAE induction and the activity phosphorylation of STAT3 and JAK2 was detected by Western blot. ( G and H ) dLN CD4 + T cells isolated from rhCTRP4-treated group or control group were expanded with irradiated autologous-presenting cells plus 10 μg/mL ( G ) or indicated concentrations ( H ) of MOG 35–55 peptide for 72 hours and subjected to cell-proliferation assay to determine T cell recall response based on BrdU assay ( G ) or quantitate the productions of IL-17A and IFN-γ ( H ). ( I ) Ctrp4 –/– female mice were immunized with MOG 35–55 peptide to induce EAE; 500 ng/mice rhCTRP4 or control (BSA) was administered intraperitoneally every day starting at disease onset on day 9 after immunization until sacrificed to restore the level of CTRP4. Clinical scores are depicted ( n = 8/group). Data are represented as mean ± SEM and are from 1 of 3 independent experiments with similar results. ( A and I ) Statistical significance was determined using 2-way repeated-measures ANOVA. Data were analyzed by unpaired Student’s t test or Mann-Whitney U test as appropriate after assessing for distribution ( C – E , G , and H ). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For pharmacological inhibition of STAT3, STAT3 inhibitor S3I-201 (Selleck, catalog S1155) was injected intraperitoneally at 5 mg/kg every 2 days.

Techniques: Control, Staining, Immunohistochemistry, Isolation, Activity Assay, Phospho-proteomics, Western Blot, Irradiation, Proliferation Assay, BrdU Staining, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: CTRP4/interleukin-6 receptor signaling ameliorates autoimmune encephalomyelitis by suppressing Th17 cell differentiation

doi: 10.1172/JCI168384

Figure Lengend Snippet: ( A ) WT mice were intraperitoneally injected with anti–IL-6R or control IgG on immunization days –1, 3, 7, 11, and 15( n = 5 mice/group). After EAE induction, mice were treated with rhCTRP4 or BSA daily from day 0 to day 27. Mean clinical scores show progression of disease. ( B ) CNS monocytes were harvested on day 18 and quantified as absolute cell numbers of indicated CNS-infiltrating cell populations gated on CD45 + . ( C ) CNS monocytes were harvested on day 18 and quantified as absolute numbers of CD4 + IFN-γ + , CD4 + IL-17A + , and CD4 + IL-17A + IFN-γ + in CNS after stimulating with PMA and inomycin with GolgiPlug for 5 hours. ( D ) Ctrp4 –/– and WT mice subjected to MOG-induced EAE were treated with selective STAT3 inhibitor S3I-201 (10 mg/kg/d dissolved in 20%DMSO/80% corn oil). Control mice were injected with equal volumes of vehicle. Each group was monitored and scored daily ( n = 7/group). ( E ) Representative images of H&E staining and LFB staining of spinal cord sections show inflammatory cell infiltration and demyelination, respectively. Scale bar: 200 μm. Data are represented as mean ± SEM and were analyzed by 1-way ANOVA with Tukey’s post test ( B and C ). Two-way repeated measures ANOVA and Holm-Šidák post hoc test ( A and D ).* P < 0.05; ** P < 0.01.

Article Snippet: For pharmacological inhibition of STAT3, STAT3 inhibitor S3I-201 (Selleck, catalog S1155) was injected intraperitoneally at 5 mg/kg every 2 days.

Techniques: Injection, Control, Staining

Journal: Pharmaceutical Biology

Article Title: Majoon Ushba alleviated IL-17A sensitized keratinocyte ferroptosis via JAK-2-STAT-3 signaling axis and reversed imiquimod induced psoriasiform inflammation

doi: 10.1080/13880209.2026.2654904

Figure Lengend Snippet: Majoon Ushba ablates the IL-17A-JAK-2/STAT-3 signaling axis. (A) Relative protein expression of JAK-2, p-JAK-2, STAT-3, and p-STAT3 in IL-17A stimulated HaCaT cells. (B) Immunofluorescence of p-STAT3 to assess nuclear translocation. (C) Colony formation assay was performed to assess the ability of the IL-17A/JAK-2-STAT-3 pathway to influence cell proliferation, magnification: 20X, Scale bar: 100μm. Data are expressed as the mean ± SEM of n = 3 independent biological replicates (separate experiments). Each dot represents a single biological replicate from one independent experiment. Mean difference and confidence intervals are shown in the Supplementary Table 1 . Statistical significance was determined by one-way ANOVA followed by Bonferroni’s post-hoc test: # ( p < 0.05), ## ( p < 0.01), and ### ( p < 0.001) versus untreated HaCaT keratinocytes; * ( p < 0.05), ** p < 0.01, and *** p < 0.001 versus IL-17A-stimulated psoriasis-like keratinocytes. Abbreviations: JAK-2, Janus kinase 2; STAT3, Signal transducer and activator of transcription 3; IL-17A, Interleukin 17 A.

Article Snippet: The STAT-3 inhibitor S3I-201 and canonical ferroptosis inhibitor (ferrostain-1) were purchased from MedChem Express (NJ, USA).

Techniques: Expressing, Immunofluorescence, Translocation Assay, Colony Assay

Journal: Pharmaceutical Biology

Article Title: Majoon Ushba alleviated IL-17A sensitized keratinocyte ferroptosis via JAK-2-STAT-3 signaling axis and reversed imiquimod induced psoriasiform inflammation

doi: 10.1080/13880209.2026.2654904

Figure Lengend Snippet: Pharmacological ablation of psoriasis pathogenic mediators by Majoon Ushba via attenuating JAK-2-STAT-3 signaling axis. (A–C) Relative gene expression of Cyr61, HMGB1, and VEGF was assessed using RT-PCR. (D–F) Relative protein expression was assessed using immunofluorescence, magnification: 20X, Scale bar: 100μm. Data are expressed as the mean ± SEM of n = 3 independent biological replicates (separate experiments). Each dot represents a single biological replicate from one independent experiment. Mean difference and confidence intervals are shown in the supplementary Table 1 . Statistical significance was determined by one-way ANOVA followed by Bonferroni’s post-hoc test: # ( p < 0.05), ## ( p < 0.01), and ### ( p < 0.001) versus untreated HaCaT keratinocytes; * ( p < 0.05), ** p < 0.01, and *** p < 0.001 versus IL-17A-stimulated psoriasis-like keratinocytes.

Article Snippet: The STAT-3 inhibitor S3I-201 and canonical ferroptosis inhibitor (ferrostain-1) were purchased from MedChem Express (NJ, USA).

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunofluorescence

Journal: Pharmaceutical Biology

Article Title: Majoon Ushba alleviated IL-17A sensitized keratinocyte ferroptosis via JAK-2-STAT-3 signaling axis and reversed imiquimod induced psoriasiform inflammation

doi: 10.1080/13880209.2026.2654904

Figure Lengend Snippet: Majoon Ushba abrogated the JAK-2-STAT-3 signaling axis in an IMQ-induced psoriasis mice model. Skin tissue from mice in the respective experimental groups was obtained following euthanasia and used to isolate total protein and RNA. (A) Relative protein levels of JAK-2, p-JAK2, STAT-3, and p-STAT3 were assessed using western blotting. (B) Additionally, the protein levels of Ki67, a cell proliferation marker, and p-STAT3 were assessed using immunohistochemistry, magnification: 20X, Scale bar: 100μm. Total RNA was used to measure the relative mRNA levels of (C) Cyr61, (D) HMGB1, and (E) VEGF by RT-PCR. The values are expressed as the mean ± SEM of n = 6 animal per group. Each data point (n = 6 per group) represents the average of three technical replicates. Mean difference and confidence intervals are shown in the Supplementary Table 1 . Statistical significance was determined by one-way ANOVA followed by Bonferroni’s post-hoc test: #p < 0.05, ##p < 0.01 and ### p < 0.01 verses control mice. * p < 0.05, ** p < 0.01 and *** p < 0.01 verses IMQ-induced psoriasis mice. Abbreviations: Cyr61, cysteine-rich angiogenic inducer 61; HMGB, High mobility group box 1; VEGF, Vascular endothelial growth factor.

Article Snippet: The STAT-3 inhibitor S3I-201 and canonical ferroptosis inhibitor (ferrostain-1) were purchased from MedChem Express (NJ, USA).

Techniques: Western Blot, Marker, Immunohistochemistry, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Pharmaceutical Biology

Article Title: Majoon Ushba alleviated IL-17A sensitized keratinocyte ferroptosis via JAK-2-STAT-3 signaling axis and reversed imiquimod induced psoriasiform inflammation

doi: 10.1080/13880209.2026.2654904

Figure Lengend Snippet: In-Silico molecular docking analysis revealed strong binding partners against IL-17RA and STAT-3. (A–C) The prospective phytoconstituents catechin, morin, and quercetin, docked against IL-17R, showed stable interactions. (D–F) The phytoconstituents, Catechin, Morin and Quercetin docked against STAT-3 showing stable interactions.

Article Snippet: The STAT-3 inhibitor S3I-201 and canonical ferroptosis inhibitor (ferrostain-1) were purchased from MedChem Express (NJ, USA).

Techniques: In Silico, Binding Assay

Journal: Nature Communications

Article Title: The metabolic hormone leptin promotes the function of T FH cells and supports vaccine responses

doi: 10.1038/s41467-021-23220-x

Figure Lengend Snippet: a B220 - CD4 + CD25 - CD44 + CD62L - CXCR5 + PD-1 + T FH cells (1 × 10 5 ) and B220 + Fas + GL-7 + GC B cells (1 × 10 5 ) from 4-Hydroxy-3-nitrophenylacetyl hapten conjugated to ovalbumin (NP-OVA) immunized WT and db/db mice (9 dpi) were co-cultured with 5 µg/mL NP-OVA for 9 days. In the db/db T FH :WT B-cell culture, IL-21 (10 ng/mL) was added. Anti-NP IgG1 titers in the supernatant were measured by ELISA (IL-21_10 ng/mL: * P = 0.0403, WT: ** P = 0.0033). b ELISA measurement of IL-21 in cultured naive CD4 + T cells from WT mice with anti-CD3/CD28 and leptin (0-200 ng/mL) stimulation for 3 days (100 ng/mL: ** P = 0.0100, 200 ng/mL: ** P = 0.0441). c Western blot showing Stat3 phosphorylation (p-Stat3) in WT naive CD4 + T cells stimulated with 200 ng/mL leptin for 2 h. Values showing the fold changes relative to the non-treated control. d Binding of Stat3 to the Il21 promoter. Stat3-ChIP assays were performed on WT naive CD4 + T cells treated with 200 ng/mL leptin for 3 h. Results showing PCR products (left) and values for the fold changes in ChIP enrichment relative to non-treated control (right) (100 ng/mL: ** P = 0.0096, 200 ng/mL: ** P = 0.0043). e Schematic of Il21 promoter construction and luciferase assay. f The transcriptional activity of the Il21 promoter. Dual-Luciferase reporter assay for the Il21 promoter in 293 T cells with 200 ng/mL leptin treatment for 12 h (100 ng/mL: ** P = 0.0082, 200 ng/mL: ** P = 0.0016). g Cd4-Cre : Stat3 +/+ or Cd4-Cre : Stat3 fl/fl naive CD4 + T cells were stimulated under IL-21-inducing conditions with 200 ng/mL leptin for 3 days. Results for IL-21 secretion showing representative FACS plots (left) and statistics (right) (nil: * P = 0.0111, Leptin: * P = 0.0144). Data are shown for individuals (dots) and mean (bars) values, and analysed by two-way ANOVA ( a ) one-way ANOVA ( b , d , f ) or Mann–Whitney U-test ( g ). * P < 0.05, ** P < 0.01. Results are representative of three independent experiments.

Article Snippet: For the intracellular staining of Bcl6 expression, naive CD4 + T cells from Cd4-Cre , Cd4-Cre:Stat3 fl/fl and Cd4-Cre:Rictor fl/fl mice were stimulated with anti-CD3 (1 μg/mL, Biolegend) and anti-CD28 (1 μg/mL, Biolegend) for 2 days, further with Stat3 inhibition (S3I-201,100 μM, Selleck Chemicals), mTOR inhibition (Rapamycin, 200 nM, Selleck Chemicals), with or without leptin (200 ng/mL) treatment for 12 h. Mouse T FH :B cells co-culture.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics, Control, Binding Assay, Luciferase, Activity Assay, Reporter Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: The metabolic hormone leptin promotes the function of T FH cells and supports vaccine responses

doi: 10.1038/s41467-021-23220-x

Figure Lengend Snippet: a Real-time PCR analysis of mouse T FH -related genes in naïve CD4 + T cells from WT mice stimulated with anti-CD3/CD28 for 2 days, further treated with leptin for 12 h ( Cxcr5 : ** P = 0.0029, Bcl6 : ** P = 0.0004, Il21 : ** P = 0.0007, Icos : ** P = 0.0028, Cd40l : ** P = 0.0023) ( n = 5). b Representative FACS plots and statistics of CXCR5 + PD-1 + T FH cells in cultured naïve CD4 + T cells from WT mice with anti-CD3/CD28 stimulation for 48 h, followed with or without leptin treatment for 12 h (Ratio: * P = 0.0200, GMFI: * P = 0.0243). c – e Western blot assay of Bcl6 with or without anti-CD3/CD28 activation ( c ), the phosphorylation of Akt, mTOR, p70S6K and S6 ( d ) and Bcl6 expressions in WT naïve CD4 + T cells with Stat3 inhibition (S3I-201, 100 μM), mTOR inhibition (Rapamycin, 200 nM) plus leptin (200 ng/mL) treatment for 12 h ( e ). Values showing the fold changes relative to the non-treated control. f Representative FACS plots and statistics of PD-1 + Bcl6 + T FH cells in cultured naïve CD4 + T cells from WT mice with anti-CD3/CD28 activation for 2 days, followed with Stat3 inhibition (S3I-201, 100 μM), mTOR inhibition (Rapamycin, 200 nM) plus leptin (200 ng/mL) treatment for 12 h (0 vs nil: ** P = 0.0004, S3I vs 0: ** P = 0.0023, Rapa vs 0: ** P = 0.0026) ( n = 5). g , h Representative FACS plots of PD-1 + Bcl6 + T FH cells in cultured naïve CD4 + T cells from Cd4-Cre:Stat3 fl/fl mice ( g ) and Cd4-Cre : Rictor fl/fl mice ( h ) with anti-CD3/CD28 activation for 2 days, followed with or without leptin (200 ng/mL) treatment for 12 h ( n = 5). Data are shown for individuals (dots) and mean (bars) values, and analysed by Mann–Whitney U-test ( a , b ), or two-way ANOVA ( f ). * P < 0.05, ** P < 0.01. Results are representative of three independent experiments.

Article Snippet: For the intracellular staining of Bcl6 expression, naive CD4 + T cells from Cd4-Cre , Cd4-Cre:Stat3 fl/fl and Cd4-Cre:Rictor fl/fl mice were stimulated with anti-CD3 (1 μg/mL, Biolegend) and anti-CD28 (1 μg/mL, Biolegend) for 2 days, further with Stat3 inhibition (S3I-201,100 μM, Selleck Chemicals), mTOR inhibition (Rapamycin, 200 nM, Selleck Chemicals), with or without leptin (200 ng/mL) treatment for 12 h. Mouse T FH :B cells co-culture.

Techniques: Real-time Polymerase Chain Reaction, Cell Culture, Western Blot, Activation Assay, Phospho-proteomics, Inhibition, Control, MANN-WHITNEY

Journal: Oncotarget

Article Title: Overcoming resistance of targeted EGFR monotherapy by inhibition of STAT3 escape pathway in soft tissue sarcoma

doi: 10.18632/oncotarget.7452

Figure Lengend Snippet: Synergistic analysis for combination therapy via CalcuSyn software

Article Snippet: EGFR inhibitor gefitinib and STAT3 inhibitor S3I-201 (NSC 74859) were purchased from Euroasian chemicals (India) and Merck (Germany), respectively.

Techniques:

Journal: Oncotarget

Article Title: Overcoming resistance of targeted EGFR monotherapy by inhibition of STAT3 escape pathway in soft tissue sarcoma

doi: 10.18632/oncotarget.7452

Figure Lengend Snippet: A. Representative images of Western blot analysis of total (t) and phosphorylated (p) STAT3 and STAT1 before and after treatment with gefitinib (10μM) and/or S3I-201 (25μM) for 24 hours in the presence of EGF on three wild type STS cell lines. B. - C. Data from at least triplicate experiments were quantified using ImageQuant software. The pSTAT3 expression in individual cell lines in the presence of EGF was normalized to β-actin and shown as percent (%) expression of post versus pre-treatment, as well as the ratio of pSTAT3/pSTAT1. D. At 24 hours post-transfection of anti-STAT3 siRNA, cells were treated with 10μM gefitinib. After incubation for 24 hours, cells were harvested for Western blot analysis. E. Representative imaging of Western blot of (from top row to bottom) cleaved C. Caspase-3, cCaspase-7 and cPARP, Cyclin D1 and Survivin before and after treatment with gefitinib and/or S3I-201 for 24 hours with EGF in 3 STS cell lines (778, 449B and HT1080). F. Data from at least triplicate experiments were quantified using ImageQuant software. All proteins with EGF stimulation were normalized to β-actin and were shown as percentage (%) expression of post/pre-treatment. G. Cells were treated with vehicle control (DMSO), gefitinib and/or S3I-201 for 24 hours and subjected to Annexin V/PI (propidium iodide) staining and flow cytometry. Error bar: standard deviation (SD). Error bar: standard deviation (SD).* p < 0.05, ** p < 0.01, *** p < 0.005.

Article Snippet: EGFR inhibitor gefitinib and STAT3 inhibitor S3I-201 (NSC 74859) were purchased from Euroasian chemicals (India) and Merck (Germany), respectively.

Techniques: Western Blot, Software, Expressing, Transfection, Incubation, Imaging, Control, Staining, Flow Cytometry, Standard Deviation

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: STAT3 promotes differentiation of monocytes to MDSCs via CD39/CD73-adenosine signal pathway in oral squamous cell carcinoma

doi: 10.1007/s00262-022-03336-9

Figure Lengend Snippet: Correlation of phosphorylated STAT3 with markers of monocytes, MDSCs and CD39/CD73 in OSCC. a Representative HE-stained images of tumor tissue and representative immunohistochemical staining of p-STAT3, CD39, CD73, CD14 and CD33 in OSCC tissue (Scale bar, 100 μm (left), 50 μm (right)). b and c The expression of p-STAT3 was significantly correlated with CD39 (p < 0.05, r = 0.29), CD73 (p < 0.05, r = 0.26), CD14 (p < 0.01, r = 0.35) and CD33 (p < 0.05, r = 0.31) in human OSCC by analyzing the tissue microarray immunohistochemical staining (n = 63). d Hierarchical clustering reveals close relation of p-STAT3, CD39, CD73, CD14 and CD33 in human OSCC (n = 63)

Article Snippet: STAT3 inhibitor S3I-201 (NSC74859) was purchased from Selleck Chemicals (501919-59-1, Westlake Village, CA) and dissolved in dimethyl sulfoxide for use at indicated concentration.

Techniques: Staining, Immunohistochemical staining, Expressing, Microarray

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: STAT3 promotes differentiation of monocytes to MDSCs via CD39/CD73-adenosine signal pathway in oral squamous cell carcinoma

doi: 10.1007/s00262-022-03336-9

Figure Lengend Snippet: S3I-201 reduces adenosine-related protein expression and inhibits CD14 monocyte differentiated into MDSCs in vitro. a Twenty-four hours after the adherent SCC7 cells were treated with S3I-201, the cell supernatant was taken for adenosine ELISA assay, and the cells were taken for western blot analysis. b Result of cck8 suggested that S3I-201 inhibited tumors at 12 h and 24 h after administration at different concentrations (100 μM, 200 μM)in vitro (n = 3, **p < 0.01, ****p < 0.0001). c and d Immunofluorescence and western blot results indicated that the expression of p-STAT3, CD39 and CD73 decreased significantly in SCC7 cells 24 h after S3I-201 treatment (Scale bar, 200 μm). e ELISA results of adenosine in the supernatant of SCC7 cells treated with different concentrations of S3I-201 (n = 3, *p < 0.05, ***p < 0.001). f and g Flow results showed that supernatants treated with S3I-201 or PSB-12379 could reduce the differentiation of CD14 monocytes into M-MDSCs, while this process can be reversed with NECA. (n = 5, **p < 0.01, ***p < 0.001, ****p < 0.0001). h Flow results showed that the cell culture medium treated with S3I-201 could more easily alleviate the immunosuppressive state of MDSCs on T cells (n = 5, *p < 0.05)

Article Snippet: STAT3 inhibitor S3I-201 (NSC74859) was purchased from Selleck Chemicals (501919-59-1, Westlake Village, CA) and dissolved in dimethyl sulfoxide for use at indicated concentration.

Techniques: Expressing, In Vitro, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Cell Culture

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: STAT3 promotes differentiation of monocytes to MDSCs via CD39/CD73-adenosine signal pathway in oral squamous cell carcinoma

doi: 10.1007/s00262-022-03336-9

Figure Lengend Snippet: S3I-201-induced STAT3 signal inhibition delays the occurrence of tumor in C3H/He mice. a C3H/He mice bearing carcinoma were treated with S3I-201 or PBS intraperitoneal (i.p) every other day for 2 weeks (n = 6). b Representative photos of mice tumor in vitro after treatment with S3I-201 or without treatment in day 14 after the first treatment. c Total tumor volume was assessed in S3I-201 and control group every two days after tumor transplanted (n = 6, ***p < 0.001). d The weight of tumor in vitro in day14 after tumor transplanted (n = 6, **p < 0.01). e The weight changes in C3H/He mice in S3I-201 or PBS group (n = 6). f Drug toxicity was assessed by body gain weight of C3H/He mice between day 1 and day 21 in each group (n = 6)

Article Snippet: STAT3 inhibitor S3I-201 (NSC74859) was purchased from Selleck Chemicals (501919-59-1, Westlake Village, CA) and dissolved in dimethyl sulfoxide for use at indicated concentration.

Techniques: Inhibition, In Vitro, Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: STAT3 promotes differentiation of monocytes to MDSCs via CD39/CD73-adenosine signal pathway in oral squamous cell carcinoma

doi: 10.1007/s00262-022-03336-9

Figure Lengend Snippet: The expression of adenosine signal pathway-related markers was significantly reduced in S3I-201 treated C3H/He mice. a and b Immunohistochemical and immunofluorescence staining indicated increased p-STAT3, CD39 and CD73 expression in control group C3H/He mice compared with S3I-201 treatment group (Scale bar, 100 μm). c Western blot analysis revealed that the protein level of p-STAT3, CD39 and CD73 were reduced with selective STAT3 activity inhibition (Scale bar, 50 μm). d ELISA results showed that adenosine concentration in tumor microenvironment decreased significantly after S3I-201 treatment (n = 3, **p < 0.01)

Article Snippet: STAT3 inhibitor S3I-201 (NSC74859) was purchased from Selleck Chemicals (501919-59-1, Westlake Village, CA) and dissolved in dimethyl sulfoxide for use at indicated concentration.

Techniques: Expressing, Immunohistochemical staining, Immunofluorescence, Staining, Control, Western Blot, Activity Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: STAT3 promotes differentiation of monocytes to MDSCs via CD39/CD73-adenosine signal pathway in oral squamous cell carcinoma

doi: 10.1007/s00262-022-03336-9

Figure Lengend Snippet: Schematic illustration: STAT3 in OSCC promotes ATP decomposition into adenosine by upregulating CD39 and CD73 expression. Then adenosine can promote the transformation of monocytes into MDSCs in the tumor microenvironment, which may eventually inhibit the expression of T cells

Article Snippet: STAT3 inhibitor S3I-201 (NSC74859) was purchased from Selleck Chemicals (501919-59-1, Westlake Village, CA) and dissolved in dimethyl sulfoxide for use at indicated concentration.

Techniques: Expressing, Transformation Assay

Journal: Mediators of Inflammation

Article Title: Ze-Qi-Tang Formula Induces Granulocytic Myeloid-Derived Suppressor Cell Apoptosis via STAT3/S100A9/Bcl-2/Caspase-3 Signaling to Prolong the Survival of Mice with Orthotopic Lung Cancer

doi: 10.1155/2021/8856326

Figure Lengend Snippet: ZQT selectively induced G-MDSC apoptosis by activating the STAT3/S100A9/Bcl-2/caspase-3 signaling pathway. (a) MDSCs from the tumor tissue of tumor-bearing mice treated with normal saline, ZQT, STAT3 inhibitor (S3I-201), or ZQT combined with S3I-201 were isolated using the MojoSort Mouse Isolation Kit. The protein expression of Ki67, STAT1, p-STAT1, STAT3, p-STAT3, S100A9, Arg-1, Bcl-2, Bax, cl-PARP, cyt c, and cl-caspase-3 in MDSCs from both normal saline and ZQT-treated mice was confirmed by Western blotting. (b) Lung tumor tissues from normal saline or ZQT-treated tumor-bearing mice were stained using S100A9, Ly6G, Ly6C, CD4, and CD8 antibodies for IHC assay (scale bar: 50 μ M). (c) T-lymphocyte proliferation assay was used to show dose-dependent suppression of T cell proliferation by G-MDSCs isolated from tumor tissue of tumor-bearing mice treated with normal saline or ZQT. CD3 + T cells isolated from tumor stained with 5 μ M CFSE were incubated with G-MDSCs for 72 h. Meanwhile, CD3 and CD28 antibodies were added for CD3 + T cell stimulation. Flow cytometry was used to quantify 72 h CFSE dilution. (d, e) G-MDSCs from tumor tissue of tumor-bearing mice treated with normal saline or ZQT were isolated using flow cytometry. The purity (CD11b + Ly6G + ) was >90% as assessed, and the protein expression of STAT3 and cl-caspase-3 was determined by IF (scale bar: 20 μ M). n = 5. Data are expressed as the mean ± SD. n/s: nonstatistical significance. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: In another experiment, 20 model mice were intragastrically administered with a daily dose of 200 μ L Ze-Qi-Tang, intraperitoneally injected with 5 mg/mL STAT3 inhibitor (S3I-201, CSNpharm), separately or combined, 3 times a week for 4 weeks.

Techniques: Saline, Isolation, Expressing, Western Blot, Staining, Lymphocyte Proliferation Assay, Incubation, Cell Stimulation, Flow Cytometry

Journal: Mediators of Inflammation

Article Title: Ze-Qi-Tang Formula Induces Granulocytic Myeloid-Derived Suppressor Cell Apoptosis via STAT3/S100A9/Bcl-2/Caspase-3 Signaling to Prolong the Survival of Mice with Orthotopic Lung Cancer

doi: 10.1155/2021/8856326

Figure Lengend Snippet: ZQT induced apoptosis in G-MDSCs in TME and inhibited its immunosuppressive activity by activating the STAT3/S100A9/Bcl-2/caspase-3 signaling pathway. Mice were treated with anti-Ly6G neutralizing antibody or isotype control by intraperitoneal injection for 14 days before surgery, after which model mice were given maintenance treatment every other day. (a, b) The bioluminescence images of model mice on day 7 and day 28 after surgery. (c) Survival curves. (d, e) The percentage of G-MDSCs, CD3 + T cells, CD4 + T cells, and CD8 + T cells in tumor tissue were analyzed by flow cytometry. (f, g) CD3 + T cells isolated from the tumor tissue of tumor-bearing C57BL/6 mice in different groups were cocultured with LLC cells for 4 h before they were incubated with CD3, CD8, and CD107 α antibodies. Events shown were finally gated on CD3 and CD8. (h) ELISA analysis for the expression of IFN- γ in cells from tumor tissue. (i) RT-qPCR analysis of Arg-1, iNOS, and S100A9 gene expression and flow cytometry for ROS production. (j) IHC staining for Ly6G and CD8 in tumor tissue (scale bar: 50 μ M). n = 5. Data are expressed as the mean ± SD. n/s: nonstatistical significance. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: In another experiment, 20 model mice were intragastrically administered with a daily dose of 200 μ L Ze-Qi-Tang, intraperitoneally injected with 5 mg/mL STAT3 inhibitor (S3I-201, CSNpharm), separately or combined, 3 times a week for 4 weeks.

Techniques: Activity Assay, Control, Injection, Flow Cytometry, Isolation, Incubation, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Gene Expression, Immunohistochemistry

Journal: Mediators of Inflammation

Article Title: Ze-Qi-Tang Formula Induces Granulocytic Myeloid-Derived Suppressor Cell Apoptosis via STAT3/S100A9/Bcl-2/Caspase-3 Signaling to Prolong the Survival of Mice with Orthotopic Lung Cancer

doi: 10.1155/2021/8856326

Figure Lengend Snippet: The immune regulatory activity of TCM formula ZQT in TME. ZQT induces the apoptosis of G-MDSCs via the STAT3/S100A9/Bcl-2/caspase-3 signaling pathway, resulting in a significant decrease in G-MDSCs and their immunosuppressive activity, consequently promoting the infiltration and killing activity of CD8 + T cells, leading to the inhibition of the proliferation of tumor cells.

Article Snippet: In another experiment, 20 model mice were intragastrically administered with a daily dose of 200 μ L Ze-Qi-Tang, intraperitoneally injected with 5 mg/mL STAT3 inhibitor (S3I-201, CSNpharm), separately or combined, 3 times a week for 4 weeks.

Techniques: Activity Assay, Inhibition