Journal: Oncotarget

Article Title: Tumor-associated macrophages promote prostate cancer migration through activation of the CCL22–CCR4 axis

doi: 10.18632/oncotarget.14185

Figure Lengend Snippet: A . Prostate cancer cells are placed in transwell inserts and treated with CCL2 (0–30 ng/ml). After 24-h incubation, PC-3 and DU145 cells that had migrated through the membrane are stained. The mean OD value is read using a microreader at 595 nm. Migration of LNCaP cells is assessed with a wound-healing assay. Data are presented as mean ± SD. B . DU145 cells are co-cultured with THP-1 and U937 cells and treated with CCL2 (30 ng/ml) for 24 h, CM is collected, and CCL17 and CCL22 levels are analyzed using ELISA. The mean OD value is read using a microreader at 450 nm, and data are presented as mean ± SD. C, D . Total RNA and protein are extracted from prostate cancer cells, and CCR2 and CCR4 gene and protein expression levels are analyzed using PCR (C) and western blot (D). E . Prostate cancer cells (1.0 × 10 5 cells/well) are seeded into 6-well plates and cultured until they reach 60%–70% confluence. Cells are incubated with anti-CCR2 or anti-CCR4 antibody and detected using a second antibody conjugated with FITC (green). Cells are counterstained with 4',6-diamidino-2-phenylindole (blue). Adjustments of brightness, contrast, and size are applied to the whole images of western blot-based analyses without elimination of any information present in the original, including backgrounds. All experiments are performed in triplicate, and mean values are shown. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant human CCL17 and CCL22 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation, Membrane, Staining, Migration, Wound Healing Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: Oncotarget

Article Title: Tumor-associated macrophages promote prostate cancer migration through activation of the CCL22–CCR4 axis

doi: 10.18632/oncotarget.14185

Figure Lengend Snippet: A . Prostate cancer cells are treated with CCL2 (30 ng/ml) for 24 h, and western blot for CCR2 is performed. B . Prostate cancer cells are treated with CCL2, CCL17, and CCL22 (30 ng/ml) for 24 h, and western blot for CCR4 is performed. C . Prostate cancer cells are co-cultured with U937 or U937-M cells for 24 h, and western blot for CCR4 is performed. D . Prostate cancer cells are placed in transwell inserts and treated with CCL17 and CCL22 (0–30 ng/ml). After 24-h incubation, PC-3 and DU145 cells that had migrated through the membrane are stained. The mean optical density (OD) value is read using a microreader at 595 nm. Migration of LNCaP cells is assessed with a wound-healing assay. Adjustments of brightness, contrast, and size are applied to the whole images of western blot-based analyses without elimination of any information present in the original, including backgrounds. Data are presented as mean ± SD. All experiments are performed in triplicate, and mean values are shown. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant human CCL17 and CCL22 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, Cell Culture, Incubation, Membrane, Staining, Migration, Wound Healing Assay

Journal: Oncotarget

Article Title: Tumor-associated macrophages promote prostate cancer migration through activation of the CCL22–CCR4 axis

doi: 10.18632/oncotarget.14185

Figure Lengend Snippet: Prostate cancer cells are incubated with a CCR2 antagonist (CCR2 ant; 20 μg/ml) or CCR4 antagonist (CCR4 ant; 10 μg/ml) for 30 min and then incubated with CCL2 (10–30 ng/ml), CCL17 (10–30 ng/ml), CCL22 (10–30 ng/ml), or CM of U937 or U937-M cells for 24 h for PC-3 and DU145 and 48 h for LNCaP. The mean optical density (OD) value is read using a microreader at 595 nm. Data are presented as mean ± SD. All experiments are performed in triplicate, and mean values are shown. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Recombinant human CCL17 and CCL22 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Incubation

Journal: Oncotarget

Article Title: Tumor-associated macrophages promote prostate cancer migration through activation of the CCL22–CCR4 axis

doi: 10.18632/oncotarget.14185

Figure Lengend Snippet: A . Phosphorylation of Akt (Ser473) proteins in prostate cancer cells is assayed using western blot at 30 min after CCL2, CCL17, or CCL22 stimulation (left panels) and after CCR4 antagonist treatment with CCL2, CCL17, or CCL22 stimulation (right panels). B . U937 cell-induced phosphorylation of Akt (Ser473) proteins in prostate cancer cells with or without CCR2 and CCR4 antagonists is also assayed using western blot. Adjustments of brightness, contrast, and size are applied to the whole images of western blot-based analyses without elimination of any information present in the original, including backgrounds. C . PC-3 cells are incubated with the Akt inhibitor AZD5363 (10 μg/ml) with CCL17 (30 ng/ml) and CCL22 (30 ng/ml) for 24 h. The mean optical density (OD) value is read using a microreader at 595 nm. Data are presented as mean ± SD. All experiments are performed in triplicate, and mean values are shown. ** p < 0.01. All experiments are performed in triplicate, and representative data are shown.

Article Snippet: Recombinant human CCL17 and CCL22 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Phospho-proteomics, Western Blot, Incubation

Journal: The World Allergy Organization Journal

Article Title: Repeated and alternate stimulations with dsRNA and SEB alter responses in macrophages and epithelial cells ☆

doi: 10.1016/j.waojou.2025.101026

Figure Lengend Snippet: Cytokine profile after repeated stimulation with dsRNA or SEB in macrophages. THP-1 derived macrophages were exposed to repeated-stimulation with varying doses of dsRNA or SEB. The cell culture supernatants were analyzed for the expression of 20 cytokines. (A) Schematic diagram illustrating cell differentiation, culture, and stimulation. (B) Heat-map analysis results of cytokine expression (excluding IL-25, IL-33, TSLP, CCL11, and CCL17). (C–H) The detailed results of cytokine expression demonstrating significant changes, IL-10 (C), CCL2 (D), CCL22 (E), CCL24 (F), CXCL10 (G), and CXCL11 (H). ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001 for comparison with its PBS control group; #p ≤ 0.05, ##p ≤ 0.01, ###p ≤ 0.001, ####p ≤ 0.0001 for comparison between each group.

Article Snippet: To compare cytokine production after repeated stimulation, 20 human cytokines, including IL-1β, IL-6, IL-10, IL-25, IL-33, TSLP, TGF-β, TNF-α, CCL1 (I-309), CCL2 (MCP-1), CCL3 (MIP-1α), CCL5 (RANTES), CCL11 (Eotaxin), CCL17 (TARC), CCL18 (PARC), CCL20 (LARC), CCL22 (MDC), CCL24 (Eotaxin-2), CXCL10 (IP-10), and CXCL11 (I-TAG), were evaluated in each culture supernatant after the second stimulation using human ELISA Duoset kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's guidelines.

Techniques: Derivative Assay, Cell Culture, Expressing, Cell Differentiation, Comparison, Control

Journal: The World Allergy Organization Journal

Article Title: Repeated and alternate stimulations with dsRNA and SEB alter responses in macrophages and epithelial cells ☆

doi: 10.1016/j.waojou.2025.101026

Figure Lengend Snippet: Cytokine profile after repeated stimulation with dsRNA or SEB in epithelial cells. Epithelial cells repeatedly underwent stimulated with different doses of dsRNA or SEB, and the cell culture supernatants were analyzed for the expression of 20 cytokines. (A) Schematic diagram illustrating cell culture and stimulation. (B) Heat-map analysis results of cytokine expression (excluding IL-25, IL-33, TSLP, CCL11, and CCL17). (C–H) The detailed results of cytokine expression highlighting significant changes, CCL5 (C), CCL24 (D), TGF-β (E), CCL2 (F), IL-6 (G), and CXCL11 (H). ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001 for comparison with its PBS control group; #p ≤ 0.05, ##p ≤ 0.01, ###p ≤ 0.001, ####p ≤ 0.0001 for comparison between each group.

Article Snippet: To compare cytokine production after repeated stimulation, 20 human cytokines, including IL-1β, IL-6, IL-10, IL-25, IL-33, TSLP, TGF-β, TNF-α, CCL1 (I-309), CCL2 (MCP-1), CCL3 (MIP-1α), CCL5 (RANTES), CCL11 (Eotaxin), CCL17 (TARC), CCL18 (PARC), CCL20 (LARC), CCL22 (MDC), CCL24 (Eotaxin-2), CXCL10 (IP-10), and CXCL11 (I-TAG), were evaluated in each culture supernatant after the second stimulation using human ELISA Duoset kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's guidelines.

Techniques: Cell Culture, Expressing, Comparison, Control

Journal: The World Allergy Organization Journal

Article Title: Repeated and alternate stimulations with dsRNA and SEB alter responses in macrophages and epithelial cells ☆

doi: 10.1016/j.waojou.2025.101026

Figure Lengend Snippet: Cytokine profile after alternate-stimulation with dsRNA and SEB in macrophages. THP-1 derived macrophages were alternately stimulated with various doses of dsRNA and SEB. The cell culture supernatants were analyzed for the expression of 20 cytokines. (A) Schematic diagram illustrating cell differentiation, culture, and stimulation. (B) Heat-map analysis results of cytokine expression (excluding IL-25, IL-33, TSLP, CCL11, and CCL17). (C–H) The detailed results of cytokine expression demonstrating significant changes, CCL5 (C), CCL20 (D), CCL22 (E), CCL24 (F), CXCL10 (G), and CXCL11 (H). ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001 for comparison with its PBS control group; #p ≤ 0.05, ##p ≤ 0.01, ###p ≤ 0.001, ####p ≤ 0.0001 for comparison between each group.

Article Snippet: To compare cytokine production after repeated stimulation, 20 human cytokines, including IL-1β, IL-6, IL-10, IL-25, IL-33, TSLP, TGF-β, TNF-α, CCL1 (I-309), CCL2 (MCP-1), CCL3 (MIP-1α), CCL5 (RANTES), CCL11 (Eotaxin), CCL17 (TARC), CCL18 (PARC), CCL20 (LARC), CCL22 (MDC), CCL24 (Eotaxin-2), CXCL10 (IP-10), and CXCL11 (I-TAG), were evaluated in each culture supernatant after the second stimulation using human ELISA Duoset kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's guidelines.

Techniques: Derivative Assay, Cell Culture, Expressing, Cell Differentiation, Comparison, Control

Journal: The World Allergy Organization Journal

Article Title: Repeated and alternate stimulations with dsRNA and SEB alter responses in macrophages and epithelial cells ☆

doi: 10.1016/j.waojou.2025.101026

Figure Lengend Snippet: Cytokine profile after alternate-stimulation with dsRNA and SEB in epithelial cells. Epithelial cells were subjected to alternate stimulation with various doses of dsRNA and SEB, and the cell culture supernatants were analyzed for the expression of 20 cytokines. (A) Schematic diagram illustrating cell differentiation and stimulation. (B) Heat-map analysis results of cytokine expression (excluding IL-25, IL-33, TSLP, CCL11, and CCL17). (C–H) The detailed results of cytokine expression demonstrating significant changes, CCL5 (C), CCL24 (D), TGF-β (E), CCL2 (F), IL-6 (G), and CXCL11(H). ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001 for comparison with its PBS control group; #p ≤ 0.05, ##p ≤ 0.01, ###p ≤ 0.001, ####p ≤ 0.0001 for comparison between each group.

Article Snippet: To compare cytokine production after repeated stimulation, 20 human cytokines, including IL-1β, IL-6, IL-10, IL-25, IL-33, TSLP, TGF-β, TNF-α, CCL1 (I-309), CCL2 (MCP-1), CCL3 (MIP-1α), CCL5 (RANTES), CCL11 (Eotaxin), CCL17 (TARC), CCL18 (PARC), CCL20 (LARC), CCL22 (MDC), CCL24 (Eotaxin-2), CXCL10 (IP-10), and CXCL11 (I-TAG), were evaluated in each culture supernatant after the second stimulation using human ELISA Duoset kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's guidelines.

Techniques: Cell Culture, Expressing, Cell Differentiation, Comparison, Control