Journal: Cell death & disease

Article Title: CCL5 derived from tumor-associated macrophages promotes prostate cancer stem cells and metastasis via activating β-catenin/STAT3 signaling.

doi: 10.1038/s41419-020-2435-y

Figure Lengend Snippet: Fig. 1 TAMs-derived CCL5 was elevated in prostate cancer and associated with metastasis. a, b The mRNA and protein expression differences of indicated genes between prostate cancer tumor tissues and para-carcinoma tissues (n = 3). c Elisa assay indicated that the blood samples of prostate cancer patients (n = 30) exhibited elevated expression of CCL5 when compared with that of healthy male participants (n = 30). d Prostate cancer patients with higher Gleason grade exhibited an increased CCL5 accumulation in the blood (n = 30). e Tissue immunofluorescence assay suggested that CCL5 and the macrophage marker CD163 were co-localized in both the primary tumor and metastatic lymph node of prostate cancer patients. Scale bar, 10 μm. f TAMs exhibited increased secretion of CCL5 than immature macrophages or prostate cancer cells. CCL5 concentrations in the cell culture supernatants of THP1-derived macrophages (M0), THP1-derived TAMs (M2), DU145 and PC3 cells were measured using Elisa method (n = 10). All values are presented as the mean ± SD. *p < 0.05, **p < 0.01.

Article Snippet: Briefly, the expression levels of CCL5, STAT3, and ALDH1A1 in prostate cancer tissue specimens were determined by the IHC method using primary antibodies against CCL5 (AF5151, Affinity), STAT3 (60199-1-lg, Proteintech) and ALDH1A1 (BF0220, Affinity).

Techniques: Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay, Marker, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Rapid Internalization and Nuclear Translocation of CCL5 and CXCL4 in Endothelial Cells

doi: 10.3390/ijms22147332

Figure Lengend Snippet: CCL5 and CXCL4 are targeted to the nucleus. ( A ) EAHy were incubated with CCL5 or CXCL4 for 60 min at 37 °C, fixed, and stained for nuclei (blue), chemokine CCL5, and CXCL4 (green). Shown are confocal micrographs. Scalebar = 10 µm. ( n = 3) ( B , C ) EAHy were incubated without (white bars) or with CCL5 ( B ) or CXCL4 ( C ) for 30 or 120 min (gray and black bars, respectively), lysed, and chemokines were determined in subcellular fractions ( n = 3, ** p < 0.01, *** p < 0.001, ANOVA with Dunn’s post-test).

Article Snippet: CCL5 was detected using a rabbit polyclonal antibody against RANTES (Abcam ab9679), and CXCL4 was detected using a rabbit polyclonal antibody against platelet factor 4 (Peprotech) at 2 μg/mL.

Techniques: Incubation, Staining

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells.

doi: 10.1210/jcem.82.5.3919

Figure Lengend Snippet: FIG. 1. RT-PCR analysis of gene transcripts. mRNA from human endometrium (lane 2), myometrium (lane 3), and endometrioma (lane 4) were reverse transcribed and amplified. Molecular mass markers are shown in lane 1. RT-PCR demonstrated a 193-bp band corre- sponding to the human RANTES mRNA in endometrium and endo- metrioma, but not in myometrium. GAPDH transcript products of 240 bp, a positive control for mRNA integrity, were detectable in all three samples. Negative controls, in which no complementary DNA (5) or no primer was added (6), revealed no amplification.

Article Snippet: The concentrated media were precleared with protein A and divided into two parts: one part was treated with VL2 antibody, and the other part was treated with a commercially available monoclonal antibody against RANTES (catalog no. MAB278, R&D Systems).

Techniques: Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Amplification, Positive Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells.

doi: 10.1210/jcem.82.5.3919

Figure Lengend Snippet: FIG. 2. RNase protection assays of RANTES and GAPDH tran- scripts. Increasing amounts of RANTES mRNA in human endome- trium of early, mid-, and late proliferative and midsecretory phases (lanes 1–4) of the menstrual cycle demonstrated by RNA protection analysis. Myometrium (lane 5) showed very low steady state levels of RANTES mRNA, whereas endometrioma (lane 6) had a high mRNA concentration. The amount of total RNA assayed was confirmed by concurrent hybridization with a GAPDH probe. The protected frag- ments for RANTES (163 bp) and GAPDH (139 bp) had the expected sizes (see Table 2).

Article Snippet: The concentrated media were precleared with protein A and divided into two parts: one part was treated with VL2 antibody, and the other part was treated with a commercially available monoclonal antibody against RANTES (catalog no. MAB278, R&D Systems).

Techniques: Concentration Assay, Hybridization

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells.

doi: 10.1210/jcem.82.5.3919

Figure Lengend Snippet: FIG. 4. RNase protection assay of human endometrial and endome- triosis cell cultures. The full-length probes for GAPDH (145 bp) and RANTES (178 bp) are shown in lanes 1 and 2, respectively. Ten micrograms of RNA were simultaneously hybridized with excess RANTES and GAPDH riboprobes, and the reaction products were subjected to RNase digestion. Hybridization with transfer RNA re- vealed no specific protected fragments (lane 3). Unstimulated endo- metriosis (lane 4) and normal endometrial stromal cells (lane 6) showed no RANTES mRNA (163 bp), but were positive for GAPDH mRNA-protected fragments (139 bp). By contrast, cytokine-stimu- lated endometriosis (lane 5) and normal endometrial stromal cells (lane 7) were positive for both RANTES and GAPDH mRNA. Endo- metrial epithelial cells (lanes 8 and 9) and Ishikawa adenocarcinoma cells (lanes 10 and 11) failed to express RANTES mRNA under basal or stimulated conditions. However, GAPDH mRNA controls were positive in these epithelial and epitheloid cells.

Article Snippet: The concentrated media were precleared with protein A and divided into two parts: one part was treated with VL2 antibody, and the other part was treated with a commercially available monoclonal antibody against RANTES (catalog no. MAB278, R&D Systems).

Techniques: Rnase Protection Assay, Hybridization

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells.

doi: 10.1210/jcem.82.5.3919

Figure Lengend Snippet: FIG. 5. Immunocytochemistry of cultured human endometrial stroma and epithelial cells. May-Gruenwald-Giemsa (A) and vimentin (B) staining served as positive controls for isolated stromal cells. These cells showed low RANTES staining under basal conditions (C), but increased RANTES expression after cytokine stimulation (D). The specificity of the immunostaining was confirmed by immunoabsorption of the primary

Article Snippet: The concentrated media were precleared with protein A and divided into two parts: one part was treated with VL2 antibody, and the other part was treated with a commercially available monoclonal antibody against RANTES (catalog no. MAB278, R&D Systems).

Techniques: Immunocytochemistry, Cell Culture, Staining, Isolation, Expressing, Immunostaining

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells.

doi: 10.1210/jcem.82.5.3919

Figure Lengend Snippet: FIG. 6. Immunochemical verification of RANTES production and se- cretion by endometrial stromal cells. Pure recombinant human RANTES (15 ng) subjected to SDS-PAGE, transferred to a Trans-Blot membrane (Bio-Rad, Richmond, CA), and reacted with anti-RANTES antibodies (MAB278) demonstrated an immunopositive band at 8 kDa (A). Endometrial stromal cells were stimulated with cytokines and metabolically labeled with [35S]cysteine and methionine. The secreted products (B) were immunoprecipitated from conditioned me- dium with VL2 (lane 1) and MAB278 (lane 2) monoclonal antibodies and separated by SDS-PAGE. These demonstrated radiolabeled bands at 8 kDa, corresponding to secreted RANTES protein.

Article Snippet: The concentrated media were precleared with protein A and divided into two parts: one part was treated with VL2 antibody, and the other part was treated with a commercially available monoclonal antibody against RANTES (catalog no. MAB278, R&D Systems).

Techniques: Recombinant, SDS Page, Membrane, Metabolic Labelling, Labeling, Immunoprecipitation, Bioprocessing

Journal: International journal of cancer

Article Title: Gastric cancer cells exploit CD4+ cell-derived CCL5 for their growth and prevention of CD8+ cell-involved tumor elimination.

doi: 10.1002/ijc.23401

Figure Lengend Snippet: FIGURE 1 – (a) Correlation between serum CCL5 level and tumor progression in patients with gastric cancer. Serum CCL5 levels in 91 patients with gastric cancer and 53 healthy volunteers were measured. Clinical stage was defined by the International Union Against Can- cer’s UICC TNM Classification of Malignant Tumors.22 All data are presented as the mean 6 SD. The number of patients in each group is displayed at the bottom of the graph. *p < 0.001 vs. healthy controls, p < 0.01 vs. T1–T2 group, p < 0.01 vs. N(2) group, §p < 0.01 vs. Stage I–II group. (b) Correlation between survival rate and serum CCL5 level in patients with gastric cancer. There was a significant dif- ference between the patients with high (>27 ng/ml) and low (27 ng/ ml) serum CCL5 levels as determined by the log-rank test (p 5 0.02). The 5-year survival rate in patients with high serum CCL5 levels was 60.4%, and in patients with low serum CCL5 levels was 86.1%. Dot- ted line: patients with high serum CCL5 levels (>27 ng/ml). Solid line: patients with low serum CCL5 levels (27 ng/ml).

Article Snippet: The sections were fixed with acetone and washed in running water for 5 min. After incubation with 3% fetal bovine serum in PBS for 10 min to block nonspecific binding, the slides were incubated with primary goat anti-human CCL5 polyclonal antibody (AF278-NA; R&D Systems, Minneapolis, MN) and a 1:80 dilution of mouse anti-human CD4 monoclonal antibody (1F6; Nichirei, Tokyo, Japan) or a 1:80 dilution of mouse anti-human CD8 monoclonal antibody (C8/144B; Nichirei) for 3 h. Sections were washed in running water for 10 min and stained with a 1:600 dilution of red fluorophore-labeled rabbit anti-goat IgG antibody (Alexa Fluor 568; Invitrogen, Carlsbad, CA) and a 1:400 dilution of green fluorophore-labeled rabbit anti-mouse IgG antibody (Alexa Fluor 488; Invitrogen) for 1 h. After washing in PBS for 10 min, sections were incubated with DAPI (4,6-diamino-2-phenylindol) to stain the nucleus.

Techniques:

Journal: International journal of cancer

Article Title: Gastric cancer cells exploit CD4+ cell-derived CCL5 for their growth and prevention of CD8+ cell-involved tumor elimination.

doi: 10.1002/ijc.23401

Figure Lengend Snippet: FIGURE 3 – CCL5 production by cocultures of whole PBMCs, CD41 cells, CD42 cells and CD81 cells with gastric cancer cell lines. Whole PBMCs, CD41 cells, CD42 cells and CD81 cells were all obtained from healthy volunteers. Cells (1 3 106) were cocultured with viable (a) or irradiated (b) gastric cancer cell lines (5 3 105), MKN45 or KATO III, for 48 h. Viable (a) or irradiated (b) MKN45 and KATO III cells were cultured for 48 h without any lymphocytes. PBMCs were also cultured alone for 48 h (a). CCL5 levels in the supernatants were measured. All data are presented as the mean 6 SE of 4 individual experiments. *p < 0.01, p < 0.05 vs. other groups.

Article Snippet: The sections were fixed with acetone and washed in running water for 5 min. After incubation with 3% fetal bovine serum in PBS for 10 min to block nonspecific binding, the slides were incubated with primary goat anti-human CCL5 polyclonal antibody (AF278-NA; R&D Systems, Minneapolis, MN) and a 1:80 dilution of mouse anti-human CD4 monoclonal antibody (1F6; Nichirei, Tokyo, Japan) or a 1:80 dilution of mouse anti-human CD8 monoclonal antibody (C8/144B; Nichirei) for 3 h. Sections were washed in running water for 10 min and stained with a 1:600 dilution of red fluorophore-labeled rabbit anti-goat IgG antibody (Alexa Fluor 568; Invitrogen, Carlsbad, CA) and a 1:400 dilution of green fluorophore-labeled rabbit anti-mouse IgG antibody (Alexa Fluor 488; Invitrogen) for 1 h. After washing in PBS for 10 min, sections were incubated with DAPI (4,6-diamino-2-phenylindol) to stain the nucleus.

Techniques: Irradiation, Cell Culture

Journal: International journal of cancer

Article Title: Gastric cancer cells exploit CD4+ cell-derived CCL5 for their growth and prevention of CD8+ cell-involved tumor elimination.

doi: 10.1002/ijc.23401

Figure Lengend Snippet: FIGURE 2 – (a) Representative immunohistochemical staining for CCL5 (3400) and CCL5 receptors (3200), CCR1, CCR3 and CCR5, on resected gastric cancer tissue. (b) Representative fluorescent double staining for CCL5/CD4 or CCL5/CD8 on resected gastric cancer tis- sue. Left column: CCL5-red fluorescence and CD4-green fluores- cence. Right column: CCL5-red fluorescence and CD8-green fluores- cence. Upper panels (3200), Lower panels (31,000).

Article Snippet: The sections were fixed with acetone and washed in running water for 5 min. After incubation with 3% fetal bovine serum in PBS for 10 min to block nonspecific binding, the slides were incubated with primary goat anti-human CCL5 polyclonal antibody (AF278-NA; R&D Systems, Minneapolis, MN) and a 1:80 dilution of mouse anti-human CD4 monoclonal antibody (1F6; Nichirei, Tokyo, Japan) or a 1:80 dilution of mouse anti-human CD8 monoclonal antibody (C8/144B; Nichirei) for 3 h. Sections were washed in running water for 10 min and stained with a 1:600 dilution of red fluorophore-labeled rabbit anti-goat IgG antibody (Alexa Fluor 568; Invitrogen, Carlsbad, CA) and a 1:400 dilution of green fluorophore-labeled rabbit anti-mouse IgG antibody (Alexa Fluor 488; Invitrogen) for 1 h. After washing in PBS for 10 min, sections were incubated with DAPI (4,6-diamino-2-phenylindol) to stain the nucleus.

Techniques: Immunohistochemical staining, Staining, Double Staining

Journal: International journal of cancer

Article Title: Gastric cancer cells exploit CD4+ cell-derived CCL5 for their growth and prevention of CD8+ cell-involved tumor elimination.

doi: 10.1002/ijc.23401

Figure Lengend Snippet: FIGURE 4 – (a) CCR1, CCR3 and CCR5 expression on the gastric cancer cell lines, MKN28 and KATO III by flowcytometric analyses. Representative data are shown from 5 individual experiments. Gray shadow indicates CCR1, 3 or 5. Solid line indicates isotype-matched controls. (b) The effect of CCL5 stimulation on cell proliferation of the gastric cancer cell lines MKN28 and KATO III. Tumor cells (1 3 104/well) were seeded in 96-well flat-bottomed plates. After 12 h, tu- mor cells were stimulated with or without indicated concentrations of rh CCL5. Incorporation of [3H] thymidine into the DNA of proliferat- ing tumor cells was measured by a liquid scintillation counter. All data are presented as the mean 6 SE of 4 individual experiments. *p < 0.05 vs. the value without CCL5.

Article Snippet: The sections were fixed with acetone and washed in running water for 5 min. After incubation with 3% fetal bovine serum in PBS for 10 min to block nonspecific binding, the slides were incubated with primary goat anti-human CCL5 polyclonal antibody (AF278-NA; R&D Systems, Minneapolis, MN) and a 1:80 dilution of mouse anti-human CD4 monoclonal antibody (1F6; Nichirei, Tokyo, Japan) or a 1:80 dilution of mouse anti-human CD8 monoclonal antibody (C8/144B; Nichirei) for 3 h. Sections were washed in running water for 10 min and stained with a 1:600 dilution of red fluorophore-labeled rabbit anti-goat IgG antibody (Alexa Fluor 568; Invitrogen, Carlsbad, CA) and a 1:400 dilution of green fluorophore-labeled rabbit anti-mouse IgG antibody (Alexa Fluor 488; Invitrogen) for 1 h. After washing in PBS for 10 min, sections were incubated with DAPI (4,6-diamino-2-phenylindol) to stain the nucleus.

Techniques: Expressing

Journal: International journal of cancer

Article Title: Gastric cancer cells exploit CD4+ cell-derived CCL5 for their growth and prevention of CD8+ cell-involved tumor elimination.

doi: 10.1002/ijc.23401

Figure Lengend Snippet: FIGURE 5 – (a) The effect of CCL5-treated cancer cells on the CD41/CD81 proportion and apoptosis of CD81 cells with cocul- tured PBMCs. Gastric cancer cells (KATO III, 5 3 105) were incu- bated with 50 ng/ml of CCL5 or medium alone for 24 h. After wash- ing cells to remove CCL5, 1 3 106 PBMCs were cocultured with CCL5-pretreated gastric cancer cells for 48 h. The CD41/CD81 pro- portion of PBMCs was examined using a flow cytometer (left column) and are shown as mean 6 SE (middle column). Upper-left quadrants indicate the percentage of CD41 cells and lower-right quadrants indi- cate the percentage of CD81 cells. Annexin V expression on CD81 cells in the cocultured PBMCs is shown (right column). Bars indicate Annexin V positive expression as determined by isotype controls. Inserted data indicate the mean percentage 6 SE of Annexin V posi- tive cells. Four individual experiments were done and the representa- tive data are depicted. Upper panel: coincubation with PBMCs and CCL5 pretreated gastric cancer cells. Lower panel: coincubation with PBMCs and untreated gastric cancer cells. (b) The effect of CCL5- treated cancer cells on the Fas/Fas Ligand expression of CD41/ CD81 cells with cocultured PBMCs. Gastric cancer cells (KATO III, 5 3 105) were incubated with 50 ng/ml of CCL5 or medium alone for 24 h. After washing cells to remove CCL5, 1 3 106 PBMCs were cocultured with CCL5-pretreated gastric cancer cells for 48 h. The CD41/CD81 proportion of PBMC was examined using a flow cy- tometer and the expression of Fas (upper) and Fas Ligand (lower) was examined. Four individual experiments were done and the representa- tive data are depicted. Solid line indicates CCL5-pretreated cells; dot- ted line indicates no treatment; dashed line indicates isotype-matched controls.

Article Snippet: The sections were fixed with acetone and washed in running water for 5 min. After incubation with 3% fetal bovine serum in PBS for 10 min to block nonspecific binding, the slides were incubated with primary goat anti-human CCL5 polyclonal antibody (AF278-NA; R&D Systems, Minneapolis, MN) and a 1:80 dilution of mouse anti-human CD4 monoclonal antibody (1F6; Nichirei, Tokyo, Japan) or a 1:80 dilution of mouse anti-human CD8 monoclonal antibody (C8/144B; Nichirei) for 3 h. Sections were washed in running water for 10 min and stained with a 1:600 dilution of red fluorophore-labeled rabbit anti-goat IgG antibody (Alexa Fluor 568; Invitrogen, Carlsbad, CA) and a 1:400 dilution of green fluorophore-labeled rabbit anti-mouse IgG antibody (Alexa Fluor 488; Invitrogen) for 1 h. After washing in PBS for 10 min, sections were incubated with DAPI (4,6-diamino-2-phenylindol) to stain the nucleus.

Techniques: Cytometry, Expressing, Incubation

Journal: International journal of cancer

Article Title: Gastric cancer cells exploit CD4+ cell-derived CCL5 for their growth and prevention of CD8+ cell-involved tumor elimination.

doi: 10.1002/ijc.23401

Figure Lengend Snippet: FIGURE 6 – The effect of neutralization of CCL5 on tumor survival rate in PBMC-bearing SCID mice. Six-week-old SCID mice were irra- diated (2 Gy, 150 kV, 5.0 mA) and 1 h later were injected i.p. with 1 3 107 KATO III cells and 1 3 107 human PBMCs. SCID mice were also injected i.p. with 1 3 107 KATO III cells alone. Tumor- and PBMC-bearing mice were given i.p. injections of 25lg/body anti- human CCL5 neutralizing antibody (n 5 10) or mouse IgG1 (n 5 10) on day 0, day 1, day 2 and day 7. The survival rates were generated using the Kaplan-Meier method, and the significance of the difference in the survival rates was determined by the log-rank test (p < 0.05).

Article Snippet: The sections were fixed with acetone and washed in running water for 5 min. After incubation with 3% fetal bovine serum in PBS for 10 min to block nonspecific binding, the slides were incubated with primary goat anti-human CCL5 polyclonal antibody (AF278-NA; R&D Systems, Minneapolis, MN) and a 1:80 dilution of mouse anti-human CD4 monoclonal antibody (1F6; Nichirei, Tokyo, Japan) or a 1:80 dilution of mouse anti-human CD8 monoclonal antibody (C8/144B; Nichirei) for 3 h. Sections were washed in running water for 10 min and stained with a 1:600 dilution of red fluorophore-labeled rabbit anti-goat IgG antibody (Alexa Fluor 568; Invitrogen, Carlsbad, CA) and a 1:400 dilution of green fluorophore-labeled rabbit anti-mouse IgG antibody (Alexa Fluor 488; Invitrogen) for 1 h. After washing in PBS for 10 min, sections were incubated with DAPI (4,6-diamino-2-phenylindol) to stain the nucleus.

Techniques: Neutralization, Injection, Generated

Journal: Nature Communications

Article Title: Migration arrest and transendothelial trafficking of human pathogenic-like Th17 cells are mediated by differentially positioned chemokines

doi: 10.1038/s41467-025-57002-6

Figure Lengend Snippet: a Culture medium collected from the upper and lower wells of transwells with HUVEC monolayers either not stimulated (NS) or stimulated with TNF-α + IFN-γ were assayed for CCL2, CCL7, and CCL8 by ELISA ( n = 3 separate experiments). Each symbol shows data from one experiment, with assays done in duplicate, and bars indicate means ± SEM. b Confocal microscopy images at × 40 magnification of permeabilized and non-permeabilized TNF-α + IFN-γ-stimulated HUVECs immunostained for CCL2, CCL7, CCL8, CCL5, CCL20, and CXCL9 (green) and stained using phalloidin for polymerized actin (magenta) and DAPI for nuclei (blue). The scale bars indicate 10 µm. Images are representative of three experiments. c Biotinylated CCL5 (0.1 μM) and CCL2 (1 μM) were incubated with CHO-K1 cells and the heparan-sulfate deficient cell line, D-677. Chemokine binding was detected using streptavidin-phycoerythrin (PE) and flow cytometry. Data shown are representative of three separate experiments.

Article Snippet: Samples were incubated at room temperature for 2 h with primary mouse antibodies against human chemokines CCL2 (5 μg, Cat# MAB679) CCL5 (5 μg, Cat# MAB278), CCL7 (5 μg, Cat# MAB282), CCL8 (5 μg, Cat# MAB281), CCL20 (5 μg, Cat# AF360), CXCL9 (5 μg, Cat# MAB392) from R&D Systems or with mouse IgG 1 isotype control (5 μg, Cat# MAB002) or mouse IgG 2B isotype control (Cat# MAB004) from R&D Systems, or with anti-human GOLPH2 (2 μg, Cat# PA5-30622) or rabbit IgG isotype control from Invitrogen, or with anti-human vWF (2 μg, Cat# ab6994) from Abcam.

Techniques: Enzyme-linked Immunosorbent Assay, Confocal Microscopy, Staining, Incubation, Binding Assay, Flow Cytometry

Journal: Nature Communications

Article Title: Migration arrest and transendothelial trafficking of human pathogenic-like Th17 cells are mediated by differentially positioned chemokines

doi: 10.1038/s41467-025-57002-6

Figure Lengend Snippet: For ( a , c ), human CD4 + T cell subgroups were isolated by FACS from the blood of healthy donors as in Supplementary Fig. and Fig. . a CCR6 +(high) CCR2 + cells were either left untreated (marked with a minus sign) or treated with CCL2 (100 ng/ml) just before and for four min after adding the cells to the flow chambers, and CCL2-treated cells were used without (marked with a minus sign) or with treatment with the CCR2 antagonist, BMS22. Data points in the left panel show numbers of cells rolling, arrested, and transmigrated, while the middle panel shows arrested cells as percentages of cells rolling and the right panel shows transmigrated cells as percentages of cells arresting. Each symbol shows data from one donor, with n = 4 individual donors in four separate experiments using CCL2. Two of these experiments included the additional treatment with BMS22. b Above is the CCL2-CXCL9 chimera sequence with signal peptide sequence shown in green, CCL2-sequence in black, and C-terminal GAG-binding sequence of CXCL9 in red. Below are confocal microscopy images of control or CCL2-CXCL9-chimera transduced, TNF-α-stimulated HUVECs immunostained for CCL2 (red) and stained with DAPI for nuclei (blue). Images are representative of three experiments. c Numbers of CCR6 +(high) CCR2 + cells, either untreated (marked with a minus sign) or treated with the CCR2 antagonist, BMS22, rolling, arrested, and transmigrated on TNF- α -activated HUVECs transduced with either control virus (marked with a minus sign) or virus encoding the CCL2-CXCL9 chimera. Middle panel shows arrested cells as percentages of cells rolling and in the right panel transmigrated cells as percentages of cells arresting. Each symbol shows data from one donor, with cells from four donors and separate experiments in each treatment group except for the BMS22-treated cells with control virus-transduced HUVECs, where cells from three donors were used. Bars indicate means ± SEM. p values were calculated using two-tailed paired Student’s t tests, and no corrections were made for multiple comparisons. Source data are provided in the file, and representative videos used to quantify rolling, arrest, and transendothelial migration are provided in Supplementary Movies – .

Article Snippet: Samples were incubated at room temperature for 2 h with primary mouse antibodies against human chemokines CCL2 (5 μg, Cat# MAB679) CCL5 (5 μg, Cat# MAB278), CCL7 (5 μg, Cat# MAB282), CCL8 (5 μg, Cat# MAB281), CCL20 (5 μg, Cat# AF360), CXCL9 (5 μg, Cat# MAB392) from R&D Systems or with mouse IgG 1 isotype control (5 μg, Cat# MAB002) or mouse IgG 2B isotype control (Cat# MAB004) from R&D Systems, or with anti-human GOLPH2 (2 μg, Cat# PA5-30622) or rabbit IgG isotype control from Invitrogen, or with anti-human vWF (2 μg, Cat# ab6994) from Abcam.

Techniques: Isolation, Sequencing, Binding Assay, Confocal Microscopy, Control, Staining, Transduction, Virus, Two Tailed Test, Migration

Journal: Nature Communications

Article Title: Migration arrest and transendothelial trafficking of human pathogenic-like Th17 cells are mediated by differentially positioned chemokines

doi: 10.1038/s41467-025-57002-6

Figure Lengend Snippet: Cytokine-activated endothelial cells upregulate expression of selectins and integrin ligands and secrete chemokines. Selectin-selectin ligand interactions help capture cells and allow them to roll along the endothelium. For pathogenic-like type 17 cells, endothelial cell-bound chemokines can then stimulate CCR6, CCR5, and CXCR3 to activate integrins for binding to intercellular adhesion molecules and mediate firm arrest. Secreted chemokines that fail to adhere to endothelial cells, such as CCL2 and other CCR2 ligands, are removed by blood flow to create a transendothelial gradient that drives TEM. Although for the sake of clarity the T cell depicted here co-expresses the four chemokine receptors, not all CCR6 +(high) CCR2 + CD4 + T cells express CCR5 and/or CXCR3.

Article Snippet: Samples were incubated at room temperature for 2 h with primary mouse antibodies against human chemokines CCL2 (5 μg, Cat# MAB679) CCL5 (5 μg, Cat# MAB278), CCL7 (5 μg, Cat# MAB282), CCL8 (5 μg, Cat# MAB281), CCL20 (5 μg, Cat# AF360), CXCL9 (5 μg, Cat# MAB392) from R&D Systems or with mouse IgG 1 isotype control (5 μg, Cat# MAB002) or mouse IgG 2B isotype control (Cat# MAB004) from R&D Systems, or with anti-human GOLPH2 (2 μg, Cat# PA5-30622) or rabbit IgG isotype control from Invitrogen, or with anti-human vWF (2 μg, Cat# ab6994) from Abcam.

Techniques: Expressing, Binding Assay

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophage-derived chemokine CCL5 facilitates the progression and immunosuppressive tumor microenvironment of clear cell renal cell carcinoma

doi: 10.7150/ijbs.74647

Figure Lengend Snippet: The overall depiction of differential expression levels of CCL5 in ccRCC and normal tissues, and its correlation with clinicopathological characteristics and outcomes in TCGA and FUSCC cohorts. ( A ) Representative immunohistochemistry images detecting CCL5 expression in paired ccRCC and normal tissues from FUSCC cohort (n=20). ( B ) Cumulative results of CCL5 IHC score from FUSCC cohort using the Students' t test. ( C ) Differential CCL5 expression in 533 tumor and 72 adjacent normal kidney samples from TCGA cohort using the Students' t test. ( D ) Distribution of paired Tumor/Normal ratio using RT-qPCR analysis from FUSCC cohort. ( E ) Western blotting assay of CCL5 expression in 4 paired fresh ccRCC and para-tumor kidney tissues. ( F ) Differential CCL5 expression in paired tumor, normal and metastatic lymph node (mlymph node) using the Students' t test (n=17). ( G ) Differential CCL5 mRNA expression with clinical AJCC stage and pathological WHO/ISUP grade using one-way ANOV test. ( H ) Multivariate Cox regression analysis enrolling clinicopathological indicators and T/N ratio in predicting overall survival displayed in a forest plot.

Article Snippet: IHC staining was performed to assess the expression levels of CCL5 using primary antibodies against CCL5 (No.36467; CST) and peroxidase-conjugated goat anti-rat IgG as previously described .

Techniques: Quantitative Proteomics, Immunohistochemistry, Expressing, Quantitative RT-PCR, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophage-derived chemokine CCL5 facilitates the progression and immunosuppressive tumor microenvironment of clear cell renal cell carcinoma

doi: 10.7150/ijbs.74647

Figure Lengend Snippet: Prognostic implications of CCL5 expression patterns in large-scale patients with ccRCC from the FUSCC and TCGA cohorts. ( A-B ) Kaplan-Meier and log-rank tests identify predictive value of CCL5 expression in prognostic prognosis in discovery set (n=290) from FUSCC cohort. ( C-D ) Kaplan-Meier and log-rank tests identify prognostic value of CCL5 expression in testing set (n=102) from FUSCC cohort. ( E-F ) Kaplan-Meier and log-rank tests identify prognostic value of CCL5 expression in validation set (n=530) from TCGA cohort.

Article Snippet: IHC staining was performed to assess the expression levels of CCL5 using primary antibodies against CCL5 (No.36467; CST) and peroxidase-conjugated goat anti-rat IgG as previously described .

Techniques: Expressing, Biomarker Discovery

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophage-derived chemokine CCL5 facilitates the progression and immunosuppressive tumor microenvironment of clear cell renal cell carcinoma

doi: 10.7150/ijbs.74647

Figure Lengend Snippet: Inhibition of CCL5 restrains proliferation, migration capacities of ccRCC cells and chemotaxis of macrophages. ( A ) Western blotting assay of CCL5 expression in transfected A498 and 786O cell lines. The data represent three independent experiments. ( B ) Cell viability of negative control and transfected ccRCC cells using CCK-8 assay. ( C ) Effect of CCL5-siRNA transfection on cell migration was determined using wound-healing assay. Histograms and Students's t test were used to evaluate the significance between two groups. ( D ) Correlation of CCL5 and epithelial-mesenchymal transition (EMT)-related markers in 533 patients with ccRCC from TCGA were detected by Spearson's correlation analysis. ( E ) The linear relationship of CCL5 expression with PI3K/AKT signaling pathway and tumor proliferation signature by Spearson's correlation. ( F ) Using Western blotting assay, expression of crucial markers in the EMT and PI3K/AKT signaling pathways were assessed.

Article Snippet: IHC staining was performed to assess the expression levels of CCL5 using primary antibodies against CCL5 (No.36467; CST) and peroxidase-conjugated goat anti-rat IgG as previously described .

Techniques: Inhibition, Migration, Chemotaxis Assay, Western Blot, Expressing, Transfection, Negative Control, CCK-8 Assay, Wound Healing Assay, Protein-Protein interactions

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophage-derived chemokine CCL5 facilitates the progression and immunosuppressive tumor microenvironment of clear cell renal cell carcinoma

doi: 10.7150/ijbs.74647

Figure Lengend Snippet: Inhibition of CCL5 restrains proliferation and chemotaxis of THP1-derived TAMs. ( A ) THP1 cells were induced differentiation into macrophages (M0) using PMA treatment and further transformed into M2 phenotype macrophages (specifically THP1-derived TAMs) induced by IL-4. ( B ) Using Western blotting assay, expression of iNOS, Arg-1, TGF-β, and CCL5 were assessed in PMA induced THP1 cells and THP1-derived TAMs. ( C ) CCK8 assay was supplemented to explore the effect of CCL5 on the proliferation ability of THP1-derived TAMs. ( D ) Representative macrophage migration images of the control group, conditioned medium (CM) of tumor cells group and the CM of CCL5-knockdown ccRCC cells. Histograms and Students's t test were used to evaluate the significance between two groups.

Article Snippet: IHC staining was performed to assess the expression levels of CCL5 using primary antibodies against CCL5 (No.36467; CST) and peroxidase-conjugated goat anti-rat IgG as previously described .

Techniques: Inhibition, Chemotaxis Assay, Derivative Assay, Transformation Assay, Western Blot, Expressing, CCK-8 Assay, Migration, Control, Knockdown

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophage-derived chemokine CCL5 facilitates the progression and immunosuppressive tumor microenvironment of clear cell renal cell carcinoma

doi: 10.7150/ijbs.74647

Figure Lengend Snippet: Perturbation of CCL5 in immune microenvironment of ccRCC and its distribution on TAMs. ( A ) Differential CCL5 expression distinguished the individual with ccRCC into two immunophenotypes using unsupervised clustering xCell algorithm for patients with ccRCC from TCGA database. ( B ) Cumulative results showing subpopulations gated on CCL5 high CD45 + leucocytes from fresh human ccRCC samples (n=20). ( C ) Representative immunohistochemistry images of CCL5 expressed macrophages. ( D ) Co-labeled with macrophage marker CD68 (red) and CCL5 (green) in ccRCC tissues. Nuclei were counterstained blue with DAPI. The right panel was merged by the aforementioned three images. ( E ) Opal multiplex immunofluorescence (mIF) was implemented to determine presence of tertiary lymphoid structures (TLS), abundance of tumor associated lymphocytes (TILs) and other cells, and PD-L1 expression in accordance to CCL5 expression on a multispectral imaging system in ccRCC tissues. ( F ) Histogram showing the aggregation of TILs and presence of TLS in ccRCC samples with different CCL5 expression levels. The bar graphs means the number of cases with TILs aggregation, immature TLS or mature TLS. The percentages written on top of each group means the proportion of this group of samples in the CCL5high or CCL5high group. ( G ) Histogram showing the percent of CD68 + CD163 + macrophage M2 in stromal and tumor samples with different CCL5 expression levels using unpaired t test. ( H ) Histogram showing the percent of PD-L1 + macrophages in ccRCC samples with different CCL5 expression levels using unpaired t test.

Article Snippet: IHC staining was performed to assess the expression levels of CCL5 using primary antibodies against CCL5 (No.36467; CST) and peroxidase-conjugated goat anti-rat IgG as previously described .

Techniques: Expressing, Immunohistochemistry, Labeling, Marker, Multiplex Assay, Immunofluorescence, Imaging

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophage-derived chemokine CCL5 facilitates the progression and immunosuppressive tumor microenvironment of clear cell renal cell carcinoma

doi: 10.7150/ijbs.74647

Figure Lengend Snippet: Intra-tumoral CCL5+ TAMs exposed a distinct subset with pro-tumorigenic exhausted features and impaired total CD8 + T cell function of ccRCC ( A ) We next evaluated the Relationship between transcriptomic CCL5 expression and cells infiltration level in the TME among 33 cancers in the TCGA database using Spearson's correlation analysis. ( B-C ) Representative images and histogram of flow cytometry showed infiltration of CCL5 high CD68 + cells in total CD45 + CD68 + cells in tumor and peritumor tissues (n=15). Data were analyzed using the Students' t test. ( D ) Differential level of immune checkpoints activation, exhaustion signatures and CXCL13 expression were compared based on Cibersort algorithm between CCL5 high and CCL5 low ccRCC samples from TCGA cohort using unpaired t test. ( E ) Differential proliferation biomarker Ki-67 expression in CD8 + T cells within CCL5 + or CCL5 - TAMs ccRCC fresh tumor tissues using flow cytometry analysis and unpaired t test. ( F ) Differential immunity stimulatory effectors in CD8 + T cells within CCL5 + or CCL5 - TAMs ccRCC fresh tumor tissues using flow cytometry analysis and unpaired t test. ( G ) Differential immune checkpoints molecules within CCL5 + or CCL5 - TAMs ccRCC fresh tumor tissues using flow cytometry analysis and unpaired t test.

Article Snippet: IHC staining was performed to assess the expression levels of CCL5 using primary antibodies against CCL5 (No.36467; CST) and peroxidase-conjugated goat anti-rat IgG as previously described .

Techniques: Cell Function Assay, Expressing, Flow Cytometry, Activation Assay, Biomarker Discovery

Journal: International Journal of Biological Sciences

Article Title: Tumor-associated macrophage-derived chemokine CCL5 facilitates the progression and immunosuppressive tumor microenvironment of clear cell renal cell carcinoma

doi: 10.7150/ijbs.74647

Figure Lengend Snippet: Accumulation of CCL5 + TAMs distinguished clinical outcomes in patients with ccRCC. ( A ) The necessary transcription signatures composing CCL5 + TAMs were enrolled in Lasso regression analysis to establish the CCL5 + TAMs model for 530 patients with ccRCC from validation TCGA cohort. ( B-C ) Kaplan-Meier and log-rank tests identify prognostic value of TAMs infiltration alone in validation set (n=530) from TCGA cohort. ( D-E ) Kaplan-Meier and log-rank tests identify prognostic value of CCL5 + TAMs infiltration in validation set (n=530) from TCGA cohort.

Article Snippet: IHC staining was performed to assess the expression levels of CCL5 using primary antibodies against CCL5 (No.36467; CST) and peroxidase-conjugated goat anti-rat IgG as previously described .

Techniques: Biomarker Discovery