Journal: Nature communications

Article Title: The HIV co-receptor CCR5 regulates osteoclast function.

doi: 10.1038/s41467-017-02368-5

Figure Lengend Snippet: Fig. 7 The blockade of CCL5 in vivo. a The serum levels of CCL3 and CCL5 in 10-week-old mice were measured by an ELISA (n = 5). b Mouse anti-CCL5 neutralizing antibodies (mCCL5 neu ab, 500 μg per mouse) were injected (once per week for 2 weeks) into 6-week-old male C57BL/6J mice (n = 5). IgG was administered to the control group. c, d μCT images (scale bars, 100 μm) and parameters (n = 5 mice per group) are shown. e Representative images of the distal femurs from the control IgG and mCCL5 ab groups. HE- (scale bars, 100 μm) and TRAP-stained sections (scale bars, 50 μm) are shown in the upper and lower panels, respectively (n = 5). f Quantitative bone histomorphometric analyses were conducted of the trabecular bones in the distal femurs of control IgG and mouse CCL5 neutralizing antibody-injected mice. *P < 0.05 (by Student’s t-test) in comparison to control and mCCL5-neuAb mice. All values are shown as the mean ± SD, n = 5

Article Snippet: Recombinant mouse CCL5 (RANTES, 478-MR) and CCL9 (MIP-1γ, 3217-MG), anti-human (MAB182), and anti-mouse (MAB-478) CCL5 neutralizing antibodies were purchased from R&D Systems.

Techniques: In Vivo, Enzyme-linked Immunosorbent Assay, Injection, Control, Staining, Comparison, Neutralizing 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) 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: Blood Advances

Article Title: Aspirin inhibits platelets from reprogramming breast tumor cells and promoting metastasis

doi: 10.1182/bloodadvances.2018026161

Figure Lengend Snippet: Platelet-derived factors selectively upregulate tumor cell IL-8. To identify factors in platelet releasate that could be driving IL-8 production in tumor cells, TRAP-, MCF-7-, and MDA-231-activated platelet releasates were analyzed using the angiogenesis array described in Figure 1. (A) Proteins identified in APR at the highest abundance were used to direct future experiments. (B) To support the array results, platelets (plt) were activated with either breast tumor cell lines (3 × 106/mL) or TRAP (5 μM), and release of CCL5 was measured by ELISA. (C) To determine whether CCL5 alone is sufficient to drive tumor cell IL-8, tumor cells were treated with 100 to 1000 ng/mL rhCCL5, and supernatant IL-8 was measured 24 hours later by ELISA. (D) IL-8 secretion in response to platelets was measured in the presence or absence of the CCL5 receptor antagonist, maraviroc (100 μM), to determine whether CCL5 is the specific factor in platelet releasate that drives tumor cell IL-8. CCR5 expression was determined by immunofluorescence in MDA-MB-231, MCF-7, BT-20, and SKBR-3 cell lines (E) and human platelets (F). (G-H) To determine whether platelet-derived CCL5 can drive metastasis, maraviroc was used to block CCR5 in invasion assays. Scale bars represent 10 μm. P values for panels B and D were determined by separate unpaired Student t tests. P values for panels C and G were determined by 1-way ANOVA, with post hoc Dunnett’s or Tukey’s multiple comparisons testing, respectively. n = 1 single experiment in panel A and 3 to 4 independent replicates per treatment group in panels B-D and G-H.

Article Snippet: Recombinant human (rh)–IL-8 and rhCCL5 were from R&D Systems (catalog no.: 208IL010, 278-RN-010).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Blocking Assay

Journal: Journal of Biological Chemistry

Article Title: Interferon Regulatory Factor 1 Is an Essential and Direct Transcriptional Activator for Interferon γ-induced RANTES/CCl5 Expression in Macrophages

doi: 10.1074/jbc.m500973200

Figure Lengend Snippet: FIG. 1. Deficiency of RANTES protein and mRNA expression in IRF-l/ mice. A, six WT and six IRF-1/ mice of 4–5 weeks of age were given injections of a single dose of MNU (50 mg/kg). One week later, the mice were sacrificed, and serum was analyzed by ELISA for mouse RANTES protein level. u.d., undetectable. Total RNA was isolated from the spleen of all of the mice and subjected to quantitative real-time PCR analysis for RANTES (B) and IRF-1 (C) expression. Data were normalized relative to GAPDH mRNA expression levels in each respective sample and further normalized to the sample from phosphate-buffered saline-treated WT mice, which was set as 100% (B) or 1 (C). D, adherent (macrophages) and nonadherent splenic cells (lymphocytes) were isolated from the entire spleen of 6–8-week-old mice and treated in vitro with MNU at different doses as indicated for 24 h, and supernatant from the cells was collected for the measurement of RANTES production by ELISA. Note that the data represent the total production of mouse RANTES (mRANTES) from all splenic macrophages and lymphocytes, respectively, normalized to 1 ml. Data represent mean S.E. of three independent experiments.

Article Snippet: Recombinant mouse RANTES protein and RANTES antibody were obtained from R&D System Inc. (Minneapolis, MN).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Isolation, Real-time Polymerase Chain Reaction, Saline, In Vitro

Journal: Journal of Biological Chemistry

Article Title: Interferon Regulatory Factor 1 Is an Essential and Direct Transcriptional Activator for Interferon γ-induced RANTES/CCl5 Expression in Macrophages

doi: 10.1074/jbc.m500973200

Figure Lengend Snippet: FIG. 2. Selective impairment of RANTES mRNA and protein synthesis in IRF-1/ macrophages. A, total RNA was isolated from WT and IRF-1/ peritoneal macrophages and subjected to RNase protection assay. 10 g of RNA was used for the expression of the indicated cytokines using mCK-5c (BD Biosciences). Data are representative of one of three separate experiments with similar results. B, kinetic expression of RANTES mRNA in IFN-treated macrophages. RNA was extracted from peritoneal macrophages of WT mice treated with IFN at different times as indicated and subjected to RT-PCR analysis for mouse RANTES mRNA expression. Mouse hypoxanthine-guanine phosphoribosyltransferase (HPRT) mRNA expression from the same samples was measured as a loading control. C, quantitative real-time PCR analysis for RANTES mRNA expression following IFN stimulation for indicated times. Data were normalized relative to GAPDH mRNA expression levels of non-IFN-treated WT cells in each respective sample. The production of RANTES was measured by ELISA from cell-free supernatants of mouse peritoneal macrophage cultures (1.0 106 cells in 1 ml) stimulated with IFN (D) and LPS (E) or primed with IFN for 16 h followed by LPS stimulation (F) for indicated times in hours. Results shown in D–F represent mean S.E. of three independent experiments.

Article Snippet: Recombinant mouse RANTES protein and RANTES antibody were obtained from R&D System Inc. (Minneapolis, MN).

Techniques: Isolation, Rnase Protection Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: Interferon Regulatory Factor 1 Is an Essential and Direct Transcriptional Activator for Interferon γ-induced RANTES/CCl5 Expression in Macrophages

doi: 10.1074/jbc.m500973200

Figure Lengend Snippet: FIG. 3. Migration of CCR5 cells is impaired in IFN-treated IRF-1/ macrophages. U87.CD4 cells stably expressing CCR5 were resuspended in complete Dulbecco’s modified Eagle’s medium, and 50 l (1 105 cells) were plated in the upper chambers. A, dose response. Increasing amounts of recombinant mouse RANTES (mRANTES) (0.25 to 10 g/ml) were aliquoted in a final volume of 29 l for each condition in the wells of the lower chamber. After a 3.5-h incubation at 37 °C, the migrated U87.CD4-CCR5 cells in the filter were stained and counted under microscope (5 fields counted/condition). B, 50 l (1 105 cells) of U87.CD4-CCR5 cells were plated in the wells of the upper chamber. Supernatant of WT mouse peritoneal macrophages treated with IFN, LPS, or IFN priming plus LPS was preincubated with an anti-RANTES antibody (a-RANTES, 15 g/ml) for 30 min at room temperature. Then 29 l of supernatant from these macrophages was applied to the individual lower chambers. Migrating cells were counted as described in A. C, 50 l (1 105 cells) of U87.CD4-CCR5 cells were plated in the wells of the upper chamber. 29 l of supernatant from WT and IRF-1/ macrophages stimulated with IFN or LPS was added to the wells in the lower chamber. Migrating cells were counted as described in A. Data represent mean S.D. of three independent experiments.

Article Snippet: Recombinant mouse RANTES protein and RANTES antibody were obtained from R&D System Inc. (Minneapolis, MN).

Techniques: Migration, Stable Transfection, Expressing, Modification, Recombinant, Incubation, Staining, Microscopy

Journal: Journal of Biological Chemistry

Article Title: Interferon Regulatory Factor 1 Is an Essential and Direct Transcriptional Activator for Interferon γ-induced RANTES/CCl5 Expression in Macrophages

doi: 10.1074/jbc.m500973200

Figure Lengend Snippet: FIG. 4. IRF-1 induces RANTES protein production and promoter activity. A, adenovirus-mediated expression of mouse IRF-l in HEK293 cells. HEK293 cells were transduced with an adenovirus expression vector containing mouse IRF-1 cDNA (IRF-1/Ad) or LacZ cDNA (LacZ/Ad) for 48–72 h. Cytoplasmic (cyto) and nuclear (nu) extracts were isolated and analyzed by Western blot using an anti-IRF-l antibody. 33 g of cytoplasmic and nuclear extracts were used in each lane. The positive control was an extract derived from peritoneal macrophages stimulated with IFN (4 h). B, WT mouse peritoneal macrophages were transduced with mouse IRF-1 cDNA or LacZ cDNA at different doses of the virus from 10 to 40 plaque-forming units (pfus)/cell for 48 h. RANTES production was measured by ELISA from the supernatants of transduced macrophages (1.0 106 cells in 1 ml). Data shown are mean S.E. of three independent experiments. C, the full-length mouse RANTES promoter-luciferase reporter construct was transiently transfected into RAW264.7 cells by electroporation. The transfected cells were stimulated with IFN, LPS, or IFN plus LPS for 7 h and the luciferase activity was measured from cell lysates. Results shown are mean S.E. from six independent experiments. D and E, the RANTES promoter-luciferase reporter construct was transiently transfected into RAW cells together with increasing amounts of an IRF-1 expression vector (D) or IRF-2 expression vector (E) or its control vector (pAct-C). Note that the IRF-1, IRF-2, or pAct-C were used in different molar ratios to the reporter, and the total amount of effector in the mixture was maintained at a constant 3.0 g with varying portions of the effector and the control vector. Luciferase activity was measured from cell lysates. Results shown are mean S.E. from three independent experiments. IRF transfected conditions versus medium condition with control vector alone: *, p 0.05; **, p 0.01; ***, p 0.001. F, kinetic expression of IRF-1 protein. RAW264.7 cells were treated with IFN for various times as indicated. 35 g of nuclear extract were used for Western blot using an anti-IRF-1 or anti-IRF-2 antibody.

Article Snippet: Recombinant mouse RANTES protein and RANTES antibody were obtained from R&D System Inc. (Minneapolis, MN).

Techniques: Activity Assay, Expressing, Transduction, Plasmid Preparation, Isolation, Western Blot, Positive Control, Derivative Assay, Virus, Enzyme-linked Immunosorbent Assay, Luciferase, Construct, Transfection, Electroporation, Control

Journal: Journal of Biological Chemistry

Article Title: Interferon Regulatory Factor 1 Is an Essential and Direct Transcriptional Activator for Interferon γ-induced RANTES/CCl5 Expression in Macrophages

doi: 10.1074/jbc.m500973200

Figure Lengend Snippet: FIG. 5. Mutagenesis of IRF1-RE in RANTES promoter. A, sequence of the WT mouse RANTES promoter region containing the IRF1-RE (underlined) and that of the ISRE mutants with specific base substitutions (boldfaced and underlined). B–D, the WT RANTES promoter-luciferase reporter construct or the mutant constructs (M1–M3) were transiently transfected into RAW264.7 cells by electroporation, together with the IRF-1 expression vector or its control vector pAct-C (B) or without cotransfection but with cellular stimulation by IFN (C) or LPS (D) for 7 h instead. Luciferase activity was measured from cell lysates and expressed as relative activity, i.e. the activity of IRF-1-cotransfected reporter over that of control vector-cotransfected reporter, which was set as 1 (B), or the activity of IFN- or LPS-stimulated cells over that of unstimulated cells, which was set as 1 (C and D, respectively). Results shown represent mean S.E. of three to four independent experiments. E, RAW264.7 cells were transiently transfected with WT or the IRF1-M1 RANTES promoter together with an expression vector for NFB p50, p65, or c-Rel as indicated. Cells were not further stimulated before luciferase activity measurement. Relative luciferase activity was calculated as fold of NFB versus its control vector (pMFG) under the medium condition. Data represent the mean S.E. from three independent experiments.

Article Snippet: Recombinant mouse RANTES protein and RANTES antibody were obtained from R&D System Inc. (Minneapolis, MN).

Techniques: Mutagenesis, Sequencing, Luciferase, Construct, Transfection, Electroporation, Expressing, Plasmid Preparation, Control, Cotransfection, Cell Stimulation, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Interferon Regulatory Factor 1 Is an Essential and Direct Transcriptional Activator for Interferon γ-induced RANTES/CCl5 Expression in Macrophages

doi: 10.1074/jbc.m500973200

Figure Lengend Snippet: FIG. 6. IRF-1 binds to RANTES promoter both in vitro and in vivo. A, nuclear extracts were isolated from thioglycolate-elicited primary mouse peritoneal macrophages from WT and IRF-1/ mice following IFN or LPS stimulation for 4 h. EMSA was performed with 10 g of nuclear extract for each sample and a double-stranded oligonucleotide probe containing the 161 to 134 region of the mouse RANTES promoter (sequence given with the critical IRF1-RE underlined). B and D, supershift EMSA was performed with the 161 to 134 probe and nuclear extracts from IFN-stimulated macrophages (B) or nuclear extract isolated from HEK293 cells transduced with the adenovirus (Ad) expressing IRF-1 or LacZ (D). A series of IRF antibodies and their control, rabbit IgG, were used. The IRF-1-related complex is indicated by an arrow. Lane 1 of A, C, and D, free probe. The asterisk in B indicates the supershifted IRF-1 complex. The question mark in D indicates a complex of an unidentified nature. E, sequence of mouse RANTES promoter containing IRF1-RE. The sequences of the pair of PCR primers used to perform ChIP are underlined and indicated as is the IRF1-RE. F, ChIP analysis was performed in WT mouse peritoneal macrophages. The amplified mouse genomic fragment derived from the endogenous RANTES promoter encompassing the IRF1-RE is indicated. The control antibody was an isotype-matched IgG. Input DNAs were used as controls. Ab, antibody; mut, mutant; mRANTES, mouse RANTES.

Article Snippet: Recombinant mouse RANTES protein and RANTES antibody were obtained from R&D System Inc. (Minneapolis, MN).

Techniques: In Vitro, In Vivo, Isolation, Sequencing, Transduction, Expressing, Control, Amplification, Derivative Assay, Mutagenesis

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