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: Cell Death & Disease

Article Title: Tsc1 expression by dendritic cells is required to preserve T-cell homeostasis and response

doi: 10.1038/cddis.2016.487

Figure Lengend Snippet: Tsc1 represses Nrp1 expression through inhibition of mTORC1-PPAR- γ pathway. ( a ) Intracellular phosphorylated Akt (Ser 473), ERK1/2 (Thr 202/204), JNK (Tyr 185), p38 MAPK (Thr 180/Tyr 182), NF- κ B p65 (Ser 536) and S6K (Ser 235/Ser 236) in splenic DCs from Tsc1 f/f and CD11c Cre Tsc1 f/f mice, treated with or without LPS ( n =3). ( b ) PPAR- γ protein was increased in splenic DCs from CD11c Cre Tsc1 f/f mice, determined by western blotting. ( c ) Nrp1 expression levels on splenic DCs after being treated with RAPA (50 ng/ml), inhibitors of mTORC1 (everolimus, 10 μ M), PPAR- γ (1 nM), STAT3 (5 μ M), HIF (10 μ M), ERK (1 μ M), JNK (10 nM) or S6K (50 nM) for 16 h ( n =3). ( d ) Intracellular phosphorylated S6K (Ser 235/Ser 236) in splenic DCs ( n =3). ( e ) Expression of Nrp1 on splenic DCs ( n =3). ( f ) Percentages of CD44 high CD62L low effector/memory cells among CD4 + Foxp3 − and CD8 + Foxp3 − T cells from the indicated mice ( n =6). All mice analyzed were 6 week old. * P <0.05, ** P <0.01; error bars represent S.D.; all data are representative of at least three independent experiments

Article Snippet: RAPA, apocynin, decylubiquinone, mTORC1 inhibitor everolimus, PPAR- γ inhibitor T0070907, STAT3 inhibitor S3I-201, HIF inhibitor 2-methoxyestradiol, ERK inhibitor FR 180204, JNK inhibitor IX and S6K inhibitor PF-4708671 were obtained from Selleck (Houston, TX, USA).

Techniques: Expressing, Inhibition, Western Blot

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: 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