Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 3×10 5 THP-1 macrophages were treated with 15 mM lactate for 24 h, and the mRNA levels of chemokines were measured by quantitative PCR. The growth medium of control macrophages was titrated to pH6.1 using sterile HCl. (B) 3×10 5 THP-1 macrophages were incubated with different concentrations of lactate for 24 h, and CCL5 gene expression was determined with quantitative PCR. (C) 10 6 THP-1 macrophages were exposed to increasing concentrations of lactate for 48 h, and the secretion of CCL5 was measured by ELISA. (D) 10 6 human primary macrophages from breast cancer patients (n=9) were cultured with different concentrations of lactate for 48 h, and CCL5 production was detected. (E) 10 6 MDA-MB-231 cells were pre-treated with 15μM GSK 2837808A for 2 h, then the media were changed, and cells were cultured for another 24 h. The conditional media (MD-231 CM) were collected and applied to 10 6 THP-1 macrophages. CCL5 concentrations were detected with ELISA. (F) Immunohistochemical staining of CD68 and CCL5 in tumor adjacent tissues (control) and breast tumors (n=28). Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Real-time Polymerase Chain Reaction, Control, Sterility, Incubation, Gene Expression, Enzyme-linked Immunosorbent Assay, Cell Culture, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 10 6 THP-1 macrophages were treated with 15 mM lactate for 48 h, and the expression of key regulators in Notch, NF-κB, STAT3 and HIF, were detected by western blot. (B) 3×10 5 THP-1 macrophages were cultured with 15 mM lactate for 24 h, and the mRNA levels of Notch ligands and receptors were measured by quantitative PCR. (C) Western blot for Notch ligands and receptors in THP-1 (10 6 ) macrophages after 48 h lactate treatment. (D) Lactate stimulated the expression of NICD in a time and dose-dependent manner. 10 6 THP-1 macrophages were treated with 15 mM lactic acid for 48 h. Data presented were representatives of at least three independent experiments. (E) 10 6 THP-1 macrophages were transfected with 50nM siNotch1, or pretreated with 50μM DAPT for 2 h, and then cultured with 15 mM lactate for 48 h. The secretion of CCL5 was measured by ELISA. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Western Blot, Cell Culture, Real-time Polymerase Chain Reaction, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 10 6 THP-1 macrophages were treated with 15 mM lactate for 72 h, and then cells were washed twice and fresh media were added. Macrophages were cultured for another 24 h and the conditional media (lactate CM) was collected. The effect of CM on breast cancer cell migration was measured by double chamber transwell assay. 5μg/ml anti-CCL5 neutralizing antibody significantly decreased lactate CM-induced cell migration. (B) 10 6 MCF-7 cells were co-cultured with 15 mM lactate-activated macrophages in the presence of 5μg/ml anti-CCL5 antibody or not, and protein levels of EMT markers were tested by western blot. (C) 10 6 breast cancer cells were co-cultured with 10 6 lactate-activated THP-1 macrophages (or 10 6 lactate-activated primary macrophages) for different time points, and the expression of CCR5 was monitored by western blot. (D) MDA-MB-231 and MCF-7 cells were transfected with shCCR5 plasmids, or pre-treated with 5μM Maraviroc for 2 h, then cell migration induced by lactate CM was detected by double chamber transwell assay. Lactate CM was described in (A). (E) MCF-7 cells (10 6 ) were transfected with pcDNA3.1-CCR5, and then cultured with 10ng/ml CCL5 for 24 h. The expression of E-cadherin, N-cadherin and vimentin was investigated by western blot. (F) 10 6 Human primary macrophages (No. 4 and No. 9) were treated with 15 mM lactate for 72 h and CM was collected as described in (A). The migration of MDA-MB-231 cells was measured in the presence of primary macrophage CM. 5μg/ml anti-CCL5 neutralizing antibody, shRNAs designed against CCR5, or 5μM Maraviroc, significantly reduced primary macrophage CM-induced cell migration. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Migration, Transwell Assay, Western Blot, Expressing, Transfection

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 3×10 5 MDA-MB-231 and MCF-7 cells were stimulated with 1-5ng/ml TGF-β1 for 24 h, and total RNA was isolated and tested for CCR5 mRNA by quantitative PCR. (B) Western blot for CCR5 protein in breast cancer cells (10 6 ) under TGF-β1 stimulation for 48 h. Data presented were representatives of at least three independent experiments. (C) MDA-MB-231 and MCF-7 cells (3×10 5 ) were co-transfected with pGL3-CCR5 and pRL-TK and exposed to different concentrations of TGF-β1 for 24 h, and luciferase activities were determined. (D) MDA-MB-231 and MCF-7 cells were pre-treated with 5μM SIS3 for 2 h, and cells were subjected to luciferase assay. (E) 10 6 MCF-7 cells were transfected with TGFβRI/ALK5 siRNA, and were then co-cultured with lactate-activated THP-1 macrophages (ratio 1:1) for 24 h. The protein levels of CCR5 were assayed by western blot. (F) The expression of TGF-β1, CCL5 and CCR5 in clinical samples obtained from breast cancer patients. The mRNA levels were measured by quantitative PCR, and the correlation between TGF-β1 and CCL5-CCR5 axis was shown. (G) Representative IHC staining for TGF-β1, CCL5 and CCR5 in breast cancer samples. The sample used was derived from 28 breast cancer cases. Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Isolation, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Luciferase, Cell Culture, Expressing, Immunohistochemistry, Derivative Assay

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) Glucose uptake, lactic acid production and ATP levels in breast cancer cells co-cultured with lactate-activated THP-1 macrophages, with or without 5μg/ml anti-CCL5 neutralizing antibody. The co-culture system was described in Figure . (B) Western blots for glycolytic enzymes in breast cancer cells treated as in (A). (C) MDA-MB-231 cells were transfected with shRNAs designed against CCR5, or pre-treated with 5μM Maraviroc for 2 h, and then subjected to cell co-culture. Glucose uptake, lactic acid production and ATP levels were measured after co-culture. The co-culture system was described in Figure . (D) The protein levels of HK2, PKM2 and LDHA in MDA-MB-231 cells cultured as in (C). (E) Recombinant human CCL5 induced aerobic glycolysis in breast cancer cells. MDA-MB-231 and MCF-7/CCR5 cells were treated with increasing concentrations of CCL5 for 12 h, and glucose uptake, lactic acid production and ATP levels were detected. (F) Western blots for glycolytic enzymes in MDA-MB-231 and MCF-7/CCR5 cells after stimulation with CCL5. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Co-Culture Assay, Western Blot, Transfection, Recombinant

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) Western blot for AMPK, c-Myc, HIF-1α and Akt in breast cancer cells co-cultured with 15mM lactic acid-activated THP-1 macrophages (ratio 1:1) for 72 h. Results presented were representatives of at least three independent experiments. (B) The expression of AMPK downstream signaling target ACC in breast cancer cells co-cultured as in (A). (C) MDA-MB-231 and MCF-7 cells were transfected with 50 nM AMPKα1 siRNA, or pretreated with 10μM compound C for 4 h, and then incubated with 15mM lactic acid-activated THP-1 macrophages (ratio 1:1) for 48 h. The glucose uptake, lactic acid production and ATP levels were detected. (D) The inhibition of AMPK abrogated macrophage-induced EMT in MCF-7 cells. Cells were treated as described in (C). After co-culture, the expression of EMT markers, E-cadherin and vimentin, was measured by western blot. (E) Recombinant human CCL5 induced the phosphorylation of AMPK in MDA-MB-231 and MCF-7/CCR5 cells. 10 6 cells were treated with 50ng/ml CCL5 for defferent time points as indicated, and phosphorylated AMPK and total AMPK were investigated by western blot. (F) Inhibition of CCR5 in MDA-MB-231 cells significantly attenuated macrophage-induced AMPK phosphorylation. MDA-MB-231 cells were transfected with shRNAs designed against CCR5, or pre-treated with 5μM Maraviroc for 2 h, then co-cultured with 15 mM lactate-activated macrophages as described in (A). After co-culture, the phosphorylation of AMPK was detected by western blot. (G) Expressions of CCL5, CCR5 and p-AMPK in samples obtained from breast cancer patients (n =28). Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, Cell Culture, Expressing, Transfection, Incubation, Inhibition, Co-Culture Assay, Recombinant, Phospho-proteomics

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) MDA-MB-231 cells were co-cultured with 15 mM lactate-activated THP-1 macrophages for 7 days, in the presence of 5μg/ml anti-CCL5 neutralizing antibody or not. MDA-MB-231 cells were then collected and injected into the tail vein of nude mice. After two weeks, animals were sacrificed and metastatic nodules on lung surfaces were counted. (B) CCR5, HK2 and p-AMPK were immunostained in MDA-MB-231 metastases. Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Injection

Journal: bioRxiv

Article Title: STING/RANTES Pathway in Airway Epithelium Stimulates Sensitization to Der p1 in an Asthma Model

doi: 10.1101/2023.07.30.550251

Figure Lengend Snippet: (A) HBEpC were cultured with cGAMP (14nM), H 2 O 2 , (100μM) or cGAMP+H 2 O 2 for 4 hours. (B-F) qPCR analysis of human IFNα (B) and IFNβ (C) and epithelial cytokines, IL-33 (D), TSLP (E), and RANTES (F). RANTES was stimulated by cGAMP and cGAMP+ H 2 O 2 . IFNβ was also stimulated by cGAMP+H 2 O 2 . Statistical analysis is performed by one-way ANOVA (means ± SEM, n=4). * p < 0.05, ** p < 0.01, **** p < 0.0001

Article Snippet: To elucidate the role of RANTES as an adjuvant during sensitization, the mice were sensitized with recombinant RANTES protein (R&D systems, Minneapolis, MN, USA, 478-MR) and 1 μg of HDM on Day 1.

Techniques: Cell Culture

Journal: bioRxiv

Article Title: STING/RANTES Pathway in Airway Epithelium Stimulates Sensitization to Der p1 in an Asthma Model

doi: 10.1101/2023.07.30.550251

Figure Lengend Snippet: (A) 8 weeks mice were treated with 20 ng of RANTES with 1 μg of HDM or 1 μg of HDM intra nasally on Day 1. Mice were challenged with 1 μg of HDM intranasally on Day 7, then lungs were extracted and analyzed on Day 8. (B) Pictures of lung sections of control and RANTES-adjuvanted, HDM-sensitized mice stained with H-E. Scale bar: 200 μm. Scale bar in picture of high magnification view: 40 μm. (C) Number of cells between bronchus and alveoli analyzed by ImageJ. Statistical analysis is performed by ordinary Mann Whitney’s U test (means ± SEM, 3 points/ section, n=5). (D) Pictures of lung sections in PBS and RANES-adjuvanted, HDM-sensitized mouse stained with PAS/Alcian blue. Scale bar: 80 μm. (E) Ratio of PAS/Alcianble area per epithelial cells (%). **** p < 0.0001

Article Snippet: To elucidate the role of RANTES as an adjuvant during sensitization, the mice were sensitized with recombinant RANTES protein (R&D systems, Minneapolis, MN, USA, 478-MR) and 1 μg of HDM on Day 1.

Techniques: Control, Staining

Journal: Journal of Neuroinflammation

Article Title: CCR5-overexpressing mesenchymal stem cells protect against experimental autoimmune uveitis: insights from single-cell transcriptome analysis

doi: 10.1186/s12974-024-03134-3

Figure Lengend Snippet: Chemokine signaling pathways were enriched in EAU retinas. ( A , B ) Chord diagrams comparing CC chemokine signaling pathway ( A ) and CXC chemokine signaling pathway ( B ) in control and EAU retinas. Edge color denotes the signaling source. Segments with large arrows represent signaling targets and inner bars represent signaling sources in which the colors indicate signaling targets. ( C ) Stacked violin plots showing the expression patterns of CC chemokine genes in control and EAU retinas based on scRNA-seq analysis. ( D ) Dot plot showing the expression of CC chemokine genes in control and EAU retinas based on bulk RNA-seq analysis. ( E ) qRT-PCR analysis showing the relative expression levels of the indicated CC chemokine genes in control and EAU retinas at 14 d.p.i. Data are presented as mean ± SD ( n = 3 mice per group). * p < 0.05, ** p < 0.01, ns, no significance. ( F ) Immunostaining of control and EAU retinal sections with an anti-CCL5 antibody with DAPI counter-labeling. Scale bar: ( F ) 20 μm

Article Snippet: Cell proliferation was then suppressed by treating the cells with the proliferation inhibitor, mitomycin C (1 μg/mL, Selleck, S8146) for 1 h. The culture-insert was removed to create gaps, followed by replacement of the old medium with fresh one with or without 100 ng/mL human CCL5 (R&D system, 278-RN).

Techniques: Protein-Protein interactions, Control, Expressing, RNA Sequencing, Quantitative RT-PCR, Immunostaining, Labeling

Journal: Journal of Neuroinflammation

Article Title: CCR5-overexpressing mesenchymal stem cells protect against experimental autoimmune uveitis: insights from single-cell transcriptome analysis

doi: 10.1186/s12974-024-03134-3

Figure Lengend Snippet: CCR5-overexpressing MSCs possess increased migrative capacity towards CCL5 in vitro and to EAU retinas in vivo. ( A ) Two-dimensional wound healing test was performed to detect the migration of MSC tdTomato and MSC CCR5 cells toward hCCL5. Shown are phase-contrast images of the cell culture at the indicated timepoints following the removal of culture-insert. ( B ) Transwell migration assay was performed to detect the migration of MSC tdTomato and MSC CCR5 cells toward hCCL5. MSCs migrated to the lower chamber surface were stained with crystal violet. ( C ) Quantification of gap closure in the wound healing test showed a significant increase in wound closure rate for MSC CCR5 cells compared to MSC tdTomato cells. Data are presented as mean ± SD ( n = 3 individual samples per group). * p < 0.01. ( D ) Quantification of migrated cells in the transwell migration assay showed a significant increase for MSC CCR5 cells compared to MSC tdTomato cells. Data are presented as mean ± SD ( n = 3 individual samples per group). * p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significance. ( E ) Schematic diagram of EAU induction and MSC transplantation into EAU mice. ( F ) Confocal images of transplanted tdTomato-positive MSCs located in the retina at 13 days post-injection. ( G ) Quantification of tdTomato-positive cells in retinas from mice transplanted with MSC CCR5 or MSC tdTomato cells. Data are presented as mean ± SD ( n = 7–10 retinas per group). * p < 0.05. d.p.i.: day(s) postimmunization; GCL: ganglion cell layer; INL: inner nuclear layer; IRBP: interphotoreceptor retinoid-binding protein; ONL: outer nuclear layer; PTX: pertussis toxin. Scale bar: (A, B) 100 μm, ( F ), 20 μm

Article Snippet: Cell proliferation was then suppressed by treating the cells with the proliferation inhibitor, mitomycin C (1 μg/mL, Selleck, S8146) for 1 h. The culture-insert was removed to create gaps, followed by replacement of the old medium with fresh one with or without 100 ng/mL human CCL5 (R&D system, 278-RN).

Techniques: In Vitro, In Vivo, Migration, Cell Culture, Transwell Migration Assay, Staining, Transplantation Assay, Injection, Binding Assay

Journal: Stem Cell Research & Therapy

Article Title: CCR5 facilitates endothelial progenitor cell recruitment and promotes the stabilization of atherosclerotic plaques in ApoE −/− mice

doi: 10.1186/s13287-015-0026-0

Figure Lengend Snippet: CCL5 expression in ascending aorta of humans and mice. (A) Immunohistochemistry of CCL5 in non-diseased artery, advanced plaques, and unstable plaques of human samples. (B) Quantitative analysis of CCL5 expression in (A) (n = 3, * P <0.05, ** P <0.01). (C) Immunohistochemistry of CCL5 in non-diseased artery, advanced plaques, and unstable plaques of ApoE −/− mice. (D) Quantitative analysis of CCL5 expression in (C) (n = 3, * P <0.05, ** P <0.01). Bars represent mean ± standard deviation. ApoE, apolipoprotein E; CCL5, chemokine (C-C motif) ligand 5; IPH, thin capped fibroatheroma with intraplaque hemorrhage (advanced unstable plaques); ND, non-diseased arteries; n.s., non-significant; TfcA, thick fibrous cap atheroma (advanced stable plaques).

Article Snippet: Recombinant mouse CCL5 (100 μg/mL; R&D Systems, Minneapolis, MN, USA), CCL5 (100 μg/mL) combined with anti-CCL5 antibody (6 μg/mL; Abcam), or EBM-2 was added into the lower chambers.

Techniques: Expressing, Immunohistochemistry, Standard Deviation

Journal: Stem Cell Research & Therapy

Article Title: CCR5 facilitates endothelial progenitor cell recruitment and promotes the stabilization of atherosclerotic plaques in ApoE −/− mice

doi: 10.1186/s13287-015-0026-0

Figure Lengend Snippet: Effects of CCL5 and CCR5 interaction on endothelial progenitor cell (EPC) migration and proliferation. (A) Transwell migration assay of EPCs after transfection with Lenti-EGFP or Lenti-CCR5. (B) Quantitative analysis of the results in (A) (n = 3, * P <0.05, ** P <0.01). (C) Representative images of 5-ethynyl-2′-deoxeuridine (EdU) cell proliferation after transfection with Lenti-EGFP or Lenti-CCR5. Arrows indicate EdU-positive cells. (D) Rate of EdU-positive cells in (C) (n = 3). Bars represent mean ± standard deviation. CCL5, chemokine (C-C motif) ligand 5; CCR5, chemokine (C-C motif) receptor 5; DAPI, 4′, 6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; n.s., non-significant.

Article Snippet: Recombinant mouse CCL5 (100 μg/mL; R&D Systems, Minneapolis, MN, USA), CCL5 (100 μg/mL) combined with anti-CCL5 antibody (6 μg/mL; Abcam), or EBM-2 was added into the lower chambers.

Techniques: Migration, Transwell Migration Assay, Transfection, Standard Deviation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cutting edge: T cells trigger CD40-dependent platelet activation and granular RANTES release: a novel pathway for immune response amplification.

doi: 10.4049/jimmunol.172.4.2011

Figure Lengend Snippet: FIGURE 3. CD40-dependent RANTES release by platelets. RANTES re- lease by soluble and membrane-bound CD40-stimulated platelets. Platelets were stimulated either with sCD40L or paraformaldehyde-fixed D1.1 cells (1: 10), and RANTES content in the supernatants was measured by ELISA. Data are mean SEM of three separate experiments. , p 0.01 for sCD40L- and D1.1 cell-stimulated platelets vs blocking Ab treatment.

Article Snippet: RANTES ELISA kits and human recombinant RANTES were purchased from R&D Systems (Minneapolis, MN) and phospho-p38 and phospho-extracellular signalregulated kinase (ERK) Abs from Cell Signaling (Beverly, MA).

Techniques: Membrane, Enzyme-linked Immunosorbent Assay, Blocking Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cutting edge: T cells trigger CD40-dependent platelet activation and granular RANTES release: a novel pathway for immune response amplification.

doi: 10.4049/jimmunol.172.4.2011

Figure Lengend Snippet: FIGURE 4. Involvement of p38 MAP kinase in platelet CD40 signaling. A, Immunoblotting of CD40-induced p38 phosphorylation in sCD40L-stimulated platelets. Platelets were stimulated with sCD40L and then lysed at the indicated time points. Immunoblotting was performed with Abs specific for phosphorylated p38 and ERK. This figure is representative of three separate experiments. B, Inhi- bition of CD40-dependent platelet activation by SB203580. Platelets were acti- vated with sCD40L with or without pretreatment with the p38 MAP kinase inhib- itor SB203580 and then submitted to flow cytometric analysis for P-selectin expression. The black curve represents the background signal from the isotype con- trol. The presence of DMSO, used for SB203580 dilution, had no effect on P- selectin expression. Data represent the mean SEM of three separate experiments. , p 0.01 for inhibitor-treated compared with sCD40L-stimulated platelets. C, Inhibition of CD40-dependent RANTES release by SB203580. Platelets were stimulated with sCD40L in the presence and absence of SB203580, and RANTES content in the supernatants was measured by ELISA. Data are mean SEM of three separate experiments. , p 0.01 for inhibitor-treated compared with sCD40L-stimulated platelets.

Article Snippet: RANTES ELISA kits and human recombinant RANTES were purchased from R&D Systems (Minneapolis, MN) and phospho-p38 and phospho-extracellular signalregulated kinase (ERK) Abs from Cell Signaling (Beverly, MA).

Techniques: Western Blot, Phospho-proteomics, Activation Assay, Inhibition, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Cutting edge: T cells trigger CD40-dependent platelet activation and granular RANTES release: a novel pathway for immune response amplification.

doi: 10.4049/jimmunol.172.4.2011

Figure Lengend Snippet: FIGURE 5. Platelet-derived RANTES mediates increased T cell recruitment. A, Detection of platelet-derived and recombinant RANTES on the surface of HIMEC by confocal microscopy. Confluent HIMEC monolayers were exposed to degranulated platelet-derived or recombinant (r) RANTES and fluorescently labeled for detection of RANTES (Alexa 488 secondary Ab, green) and nuclei (DAPI, blue). This figure is representative of four separate experiments. B, Platelet-derived RANTES-mediated T cell adhesion to HIMEC and inhibition by met-RANTES. HIMEC monolayers were left untreated (baseline) or exposed to degranulated platelet-derived or recombinant RANTES. MOLT4 cells, preincubated or not with met-RANTES, were added to the HIMEC monolayers. The number of adherent cells in each experimental condition was expressed as mean SEM of four separate experiments. , p 0.05 for met-RANTES-pretreated compared with untreated MOLT4 cells.

Article Snippet: RANTES ELISA kits and human recombinant RANTES were purchased from R&D Systems (Minneapolis, MN) and phospho-p38 and phospho-extracellular signalregulated kinase (ERK) Abs from Cell Signaling (Beverly, MA).

Techniques: Derivative Assay, Recombinant, Confocal Microscopy, Labeling, Inhibition