Journal: The Journal of Immunology

Article Title: Response to Comment on “Decoy Receptor 3 Expression in AsPC-1 Human Pancreatic Adenocarcinoma Cells via the Phosphatidylinositol 3-Kinase-, Akt-, and NF-κB-Dependent Pathway”

doi: 10.4049/jimmunol.0990054

Figure Lengend Snippet: FIGURE 1. ELISA and immunoprecipitation for determination of DcR3 protein expression in the human pancreatic cancer cell lines AsPC-1 and PANC-1. A, Cells (1 105) were cultured in 24-well plates, the supernatants were collected after 48 h of incubation, DcR3 levels were detected by ELISA, and the cells were collected to quantify that the protein concentration was equal. B, Equal amounts of concentrated medium from cells was immunopre- cipitated (IP) with 1 g of anti-DcR3 Ab, followed by Western blotting (WB) using the same DcR3 Ab. The GAPDH Ab was used for loading control. HT-29 cells were used as the negative control and SW480 cells were used as positive control for DcR3 expression, respectively. The blots shown are the representative of those obtained in three separate experiments. The data are the mean SEM for three separate experiments.

Article Snippet: We used a commercial DuoSet ELISA development system (catalog number DY142; R&D Systems) to detect DcR3 expression levels, and all of our results were comprehended within the detection range of this kit, which limited the analytic concentration below 12 ng/ml in accordance with the manufacturer’s suggestion.

Techniques: Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Expressing, Cell Culture, Incubation, Protein Concentration, Western Blot, Control, Negative Control, Positive Control

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The effect and mechanism of TIPE in promoting M2 polarization through DcR3 in the microenvironment of colorectal cancer

doi: 10.1007/s00262-026-04531-8

Figure Lengend Snippet: TIPE promotes M2 polarization through DcR3. A GEPIA database analysis of the correlation between TIPE and M2 marker molecules CD206, CD163, and IL-10 in CRC tissues. B Western blot verified the transfection efficiency of TIPE overexpression and knockdown plasmids in SW480 and HCT116, respectively. C and D Expression of CD206, CD163, IL-10, CD80, CD86, and IL-1β was detected by qPCR after the action of SW480-OE, and SW480-V CM on murine-derived macrophages RAW264.7. E UALCAN database analysis of DcR3 expression in CRC in situ tumors. F Expression and correlation analysis of DcR3 and TIPE in tumors and paracancerous tissues of CRC patients were detected by qPCR. G Western blot validation of DcR3 expression in TIPE differentially expressing CRC cells. H ELISA for DcR3 expression in SW480-OE, SW480-V supernatants. I GEO database analysis of DcR3, CD163 expression in CRC tissues. J GEPIA database analysis of DcR3 correlation with CD206, CD163, and IL-10 in CRC tissues. K Measurement of ARG-1 enzymatic activity in M0 macrophages following exogenous DcR3 treatment. L Expression of CD206, CD163, ARG-1, and IL-10 after exogenous DcR3 action on M0 was detected by qPCR. M Proportion of CD206, CD163, and ARG-1-positive macrophages after exogenous DcR3 action on M0 was detected by flow cytometry. N The proportion of CD206, CD163, and ARG-1-positive macrophages was detected by flow cytometry after addition of exogenous DcR3 and anti-DcR3 to SW480-V and SW480-OE CM-acting M0, respectively. Data were drawn as mean ± SD (n ≥ 3). * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: ELISA was performed using the DcR3 enzyme-linked immunosorbent assay kit (RayBiotech, USA) according to the manufacturer’s protocol.

Techniques: Analysis, Marker, Western Blot, Transfection, Over Expression, Knockdown, Expressing, Derivative Assay, In Situ, Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Activity Assay, Flow Cytometry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The effect and mechanism of TIPE in promoting M2 polarization through DcR3 in the microenvironment of colorectal cancer

doi: 10.1007/s00262-026-04531-8

Figure Lengend Snippet: DcR3 exerts its function via binding to HSPG2 in an HS-dependent manner. A KEGG pathway enrichment in RNA-Seq sequencing results. B The volcano plot represented the expression of HSPG2 in the RNA-Seq sequencing results. C qPCR detection of HSPG2 expression in tumors and paracancerous tissues of CRC patients and analysis of correlation with TIPE. D qPCR was performed to detect the expression of HSPG2 after SW480-OE, SW480-V, HCT116-V, and HCT116-KD CM action on M0. E Western blot detection of HSPG2 expression after SW480-OE, SW480-V CM-acting M0. F Endotoxin of exogenous DcR3 was measured via the turbidimetric limulus test. DcR3 was prepared at a concentration of 0.55 mg/mL, and applied at 5.5 μg/mL. G qPCR detection of HSPG2 expression after exogenous DcR3 acting M0. H Western blot detection of HSPG2 expression after addition of exogenous DcR3, anti-DcR3 to SW480-V, SW480-OE CM-acting M0, respectively. I PyMOL predicts DcR3 and HSPG2 binding sites. J Flow cytometry was used to detect the surface expression of HSPG2. K ELISA was used to detect DcR3 levels in cell culture supernatants. Samples were collected from M0 macrophages treated with DcR3 for 24 h, with or without heparin sodium (HS) or heparinase III pretreatment. L Western blot was used to detect p-PI3K and p-AKT levels. M0 macrophages were treated with heparin sodium (HS) or heparinase III, followed by incubation with DcR3 for 24 h. M Immunofluorescence detection of the colocalization between DcR3 and HSPG2. Data were drawn as mean ± SD (n ≥ 3). * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: ELISA was performed using the DcR3 enzyme-linked immunosorbent assay kit (RayBiotech, USA) according to the manufacturer’s protocol.

Techniques: Binding Assay, RNA Sequencing, Sequencing, Expressing, Analysis, Western Blot, Concentration Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Incubation, Immunofluorescence

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The effect and mechanism of TIPE in promoting M2 polarization through DcR3 in the microenvironment of colorectal cancer

doi: 10.1007/s00262-026-04531-8

Figure Lengend Snippet: DcR3–HSPG2 complex activates PI3K, followed by the recruitment and activation of AKT1. A KEGG pathway enrichment in RNA-Seq sequencing results. B The volcano plot represented the expression of AKT1 in the RNA-Seq sequencing results. C Western blot was used to detect the expression of AKT1 and p-AKT1 after SW480-OE and SW480-V CM action on M0. D qPCR detection of AKT1 expression after exogenous DcR3 action on M0. E Western blot detected the expression of AKT1 and p-AKT1 after addition of exogenous DcR3 and anti-DcR3 to SW480-V and SW480-OE CM following acting on M0, respectively. F The GEPIA database was analyzed for HSPG2 and AKT1 correlation. G After applying siRNA to silence HSPG2 expression, qPCR was performed to detect AKT1 expression. H Western blot analysis of AKT1, p-AKT1, PI3K, and p-PI3K expression following siRNA-mediated silencing of HSPG2. I Membrane protein extraction and Western blot detection of Na⁺/K⁺-ATPase and AKT1 in HSPG2-silenced macrophages stimulated with exogenous DcR3. J Western blot analysis of AKT1, p-AKT1, PI3K, and p-PI3K expression in macrophages pretreated with the PI3K-specific inhibitor LY294002. K PyMOL predicts potential interaction regions between HSPG2 and AKT1. L Co-immunoprecipitation (Co-IP) assay demonstrating the physical interaction between HSPG2 and AKT1. M Immunofluorescence assay showing the colocalization of DcR3 and HSPG2. Data were drawn as mean ± SD (n ≥ 3). * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: ELISA was performed using the DcR3 enzyme-linked immunosorbent assay kit (RayBiotech, USA) according to the manufacturer’s protocol.

Techniques: Activation Assay, RNA Sequencing, Sequencing, Expressing, Western Blot, Analysis, Membrane, Protein Extraction, Co-Immunoprecipitation Assay, Immunofluorescence

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The effect and mechanism of TIPE in promoting M2 polarization through DcR3 in the microenvironment of colorectal cancer

doi: 10.1007/s00262-026-04531-8

Figure Lengend Snippet: TIPE promotes macrophage proliferation and migration via DcR3. A SW480, HCT116 CM were all mixed with DcR3 and anti-DcR3 to act on Mφ, respectively, and CCK-8 detected the proliferation of THP-1 ( B ) Scratch wound assay detecting RAW264.7 cell migration. Macrophages were treated with SW480 or HCT116 conditioned medium (CM) supplemented with exogenous DcR3 or anti-DcR3, respectively. C Transwell detection of RAW264.7 infiltration after SW480-V, SW480-OE, and exogenous DcR3 alone act on Mφ. Data were drawn as mean ± SD (n ≥ 3). * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: ELISA was performed using the DcR3 enzyme-linked immunosorbent assay kit (RayBiotech, USA) according to the manufacturer’s protocol.

Techniques: Migration, CCK-8 Assay, Scratch Wound Assay Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The effect and mechanism of TIPE in promoting M2 polarization through DcR3 in the microenvironment of colorectal cancer

doi: 10.1007/s00262-026-04531-8

Figure Lengend Snippet: M2 promotes CRC proliferation and migration. A GEPIA database analysis of the correlation between TIPE, DcR3 and CXCL8 in CRC tissues. B qPCR was performed to detect the expression of CXCL8 after SW480-OE, SW480-V CM and exogenous DcR3 acting on M0. C and D Kaplan–Meier plotter database to analyze the relationship between the expression of CD206, CD163, ARG-1, IL-10, HSPG2, and AKT1 and the prognosis of CRC patients. E and F The effect of M2 CM on SW480-OE proliferation was detected by live cell counting and CCK-8. G Transwell migration assay detecting the regulatory effect of M2 macrophages on SW480-OE cell migration. H Scratch experiments were performed to detect the effect of M2 CM on SW480-OE migration. I qPCR was performed to detect CXCR1/2 expression in SW480-OE, SW480-V. J qPCR detection of CXCR1/2 expression in SW480-OE after siRNA-CXCR1/2 transfection. K Cell counting and CCK-8 assays detected the effect of M2 CM on SW480-OE proliferation after siRNA-CXCR1/2 transfection. L Transwell migration assay detecting the effect of M2 macrophages on the migration of SW480-OE cells transfected with siRNA-CXCR1/2. M Scratch assay to detect the effect of M2 CM on SW480-OE migration after siRNA-CXCR1/2 transfection. Data were drawn as mean ± SD (n ≥ 3). * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: ELISA was performed using the DcR3 enzyme-linked immunosorbent assay kit (RayBiotech, USA) according to the manufacturer’s protocol.

Techniques: Migration, Analysis, Expressing, Cell Counting, CCK-8 Assay, Transwell Migration Assay, Transfection, Scratch Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: The effect and mechanism of TIPE in promoting M2 polarization through DcR3 in the microenvironment of colorectal cancer

doi: 10.1007/s00262-026-04531-8

Figure Lengend Snippet: In vivo experiments have confirmed that TIPE promotes M2 polarization and CRC proliferation through DcR3. A Schematic diagram of nude mouse inoculation. B Changes in body weight of mice. C Tumor volume statistics. D IHC detection of Ki-67 expression in CRC tumor tissues. E – G Proportion of CD86, CD206, CD163, and ARG-1-positive macrophages in tumor tissues, paraneoplastic lymph nodes, and spleen were detected by flow cytometry. H Expression of HSPG2, AKT1, and CXCL8 in CRC tumor tissues was detected by qPCR. I Western blot detection of HSPG2, AKT1, p-AKT1 expression in CRC tumor tissues. J IHC detection of p-AKT1 expression in CRC tumor tissues. Data were drawn as mean ± SD (n ≥ 3). * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: ELISA was performed using the DcR3 enzyme-linked immunosorbent assay kit (RayBiotech, USA) according to the manufacturer’s protocol.

Techniques: In Vivo, Expressing, Flow Cytometry, Western Blot