protein expression  (R&D Systems)

Journal: Science advances

Article Title: Caspase-1-dependent pyroptosis converts αSMA + CAFs into collagen-III high iCAFs to fuel chemoresistant cancer stem cells.

doi: 10.1126/sciadv.adt8697

Figure Lengend Snippet: Fig. 4. Tumor cell pyroptosis skews CAFs toward iCAFs in a CCR6-dependent mechanism. (A) Representative image illustrating a cytokine protein array (i.e., Proteome Profiler Human XL Cytokine Array Kit, R&D Systems) probed with the supernatant collected from WT and Casp1 KO T24 cancer cells after 48 hours of gemcitabine treat- ment. Red, blue, and green boxes highlighting the individual cytokines or chemokines with the most differential expression (i.e., reduction) in Casp1 KO versus WT Gem- treated supernatant. (B) Bar graph quantifying the intensity of three cytokines differentially released in WT and Casp1 KO supernatant, i.e., CXCL5, CXCL10, and CCL20. (C) ELISA quantification of CXCL5, CXCL10, and CCL20 protein concentration in the supernatants collected from gemcitabine-treated WT and Casp1 KO T24 blad- der cancer cells. (D) Flow cytometry assessing the percentage of αSMAhigh/+ CAFs and αSMA−PDGFβ+ iCAFs treated with CM from Gem-treated T24 cells with anti-CXCR2 neutralizing Ab, (anti-CXCR2 Ab, blocking receptor downstream to CXCL5), anti-CXCL10 neutralizing Ab, anti-CCL20 neutralizing ab, and CCR6i (blocking receptor down- stream to CCL20). (E) Violin dot plot quantifying the relative changes in the percentage of αSMA−PDGFβ+ iCAFs upon CM-Gem ± chemokine or chemokine receptor neutralizing Ab treatments.

Article Snippet: Tumor cell pyroptosis skews CAFs toward iCAFs in a CCR6- dependent mechanism. (A) Representative image illustrating a cytokine protein array (i.e., Proteome Profiler human Xl cytokine Array Kit, R&d Systems) probed with the supernatant collected from Wt and casp1 KO t24 cancer cells after 48 hours of gemcitabine treatment.

Techniques: Protein Array, Quantitative Proteomics, Enzyme-linked Immunosorbent Assay, Protein Concentration, Flow Cytometry, Blocking Assay

Journal: Frontiers in Immunology

Article Title: E2F1-induced autocrine IL-6 inflammatory loop mediates cancer-immune crosstalk that predicts T cell phenotype switching and therapeutic responsiveness

doi: 10.3389/fimmu.2024.1470368

Figure Lengend Snippet: The impact of E2F1 on immune cell infiltration and cytokine release in coculture with T cells. (A, B) Correlation of E2F1 expression with infiltration of monocytes, eosinophils, and neutrophils in melanoma patients with primary tumors (A) or metastasis (B) . (C, D) Estimation of CD4 + Th1 and Th2 cell infiltration cells in melanoma patients with primary tumors or metastasis correlated to the expression of E2F1, IL-6 (C) , and STAT3, CD274 (D) . Significant correlations are highlighted in red. (E, F) Cytokine assays of supernatants from different culture/coculture conditions were compared as illustrated showing an E2F1-dependent increased secretion of immunosuppressive type 2 cytokines (E) that was completely reversed after E2F1-KD in melanoma cells (F) . Asterisks indicate transcriptionally upregulated factors. FC, fold change.

Article Snippet: To measure secreted proteins Proteome Profiler Human Cytokine Array Kit was used according to manufacturer’s instruction (#ARY005B, R&D Systems).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: E2F1-induced autocrine IL-6 inflammatory loop mediates cancer-immune crosstalk that predicts T cell phenotype switching and therapeutic responsiveness

doi: 10.3389/fimmu.2024.1470368

Figure Lengend Snippet: Mathematical model and overview of the melanoma-CD4 + T cell crosstalk with E2F1-STAT3/IL-6/IL6R axis. (A) Mathematical model of the E2F1-STAT3/IL-6 axis including the auto- and paracrine feedback loops from melanoma and immune cells. The heatmap shows that factors related to the IL6R-STAT3-E2F1 pathway are expressed in melanoma and CD4 + T cells prior to activation and crosstalk. (B) Crosstalk simulation illustrating IL-6 expression in melanoma and CD4 + T cells. (C) Output of simulation representing expression levels of E2F1 or STAT3 that are sufficient to trigger IL-6 secretion in melanoma cells. (D) The impact from E2F1 on the melanoma T cell crosstalk where high E2F1 expression leads to an immunosuppressive TME through feedback loops triggered increased release of IL-6 inducing a Th2 shift (left). In contrast, E2F1 depletion in melanoma cells blocks the augmented IL-6 secretion and, in turn, diminishes type-2 cytokine production favoring an anti-tumor Th1 response.

Article Snippet: To measure secreted proteins Proteome Profiler Human Cytokine Array Kit was used according to manufacturer’s instruction (#ARY005B, R&D Systems).

Techniques: Activation Assay, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Gasdermin-E Mediated Pyroptosis—A Novel Mechanism Regulating Migration, Invasion and Release of Inflammatory Cytokines in Rheumatoid Arthritis Fibroblast-like Synoviocytes

doi: 10.3389/fcell.2021.810635

Figure Lengend Snippet: Alleviation of IL-6 and other inflammatory cytokines release by downregulation of GSDME-mediated RA-FLSs pyroptosis. (A,C) After siRNA transfection and TNF-α + hypoxia treatment, cell culture supernatant was collected and the levels of cytokines and chemokines were measured using Proteome Profiler Human Cytokine Array Kit from R&D systems (A,B) . Supernatant levels of IL-6, IL-8 were further verified by ELISA (n = 3) (C) . (D,E) HE and IHC staining for IL-6 of synovium tissues from RA ( n = 21), OA ( n = 4) and Orth.A ( n = 5) patients (scale bar = 100 μm). RA synovium tissues were futher divide into low GSDME-NT level ( n = 6) and high GSDME-NT level ( n = 15) groups based on matched GSDME-NT level determined by western blotting. Data are shown as mean ± s. e.m and results are representative of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 using one-way ANOVA.

Article Snippet: Supernatants of cells with the indicated treatments were collected and centrifuged (12,000 rpm, 5 min), and secretion of cytokines was analyzed by a Protein Profiler Human Cytokine Array Kit (ARY005B, R&D Systems, United States) following the manufacturer’s protocol.

Techniques: Transfection, Cell Culture, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Western Blot