met ccl5  (R&D Systems)

Journal: iScience

Article Title: Derived myeloid lineage induced pluripotent stem as a platform to study human C-C chemokine receptor type 5Δ32 homozygotes

doi: 10.1016/j.isci.2023.108331

Figure Lengend Snippet:

Article Snippet: In the lower part of the transwell, DMEM medium (Lonza) was added with recombinant–soluble human RANTES (rh-RANTES; R&D) that was used as an attractant chemokine at different concentrations (1, 3, and 10 ng/ml).

Techniques: Virus, Recombinant, Modification, Purification, Blocking Assay, Luminex, Pore Size, Amplification, Software

Journal: Frontiers in Cardiovascular Medicine

Article Title: Sox13 is a novel flow-sensitive transcription factor that prevents inflammation by repressing chemokine expression in endothelial cells

doi: 10.3389/fcvm.2022.979745

Figure Lengend Snippet: Monocyte adhesion induced by siSOX13 is prevented by treating HAECs with MetCCL5. (A) HAECs overexpressing SOX13 or RFP control were exposed to OSS or ULS for another 24 h. Following shear, THP1 monocytes were added to the conditioned media (CM), and adhered monocytes were counted. (B) HAECs treated with siSOX13 or siCtrl were exposed to OSS or ULS for another 24 h. Following shear, THP1 monocytes were added to the CM, and adhered monocytes were counted. (C,D) HAECs treated with siSOX13 or siCtrl were exposed to OSS or ULS in the presence or absence of MetCCL5 for another 24 h. Following shear, THP1 monocytes were added to the CM, and adhered monocytes were counted (C) . In panel (D) , following shear, CM was removed, HAECs were washed with fresh medium, THP1 monocytes were added to the CM, and adhered monocytes were counted. Mean±SEM are shown, n = 3 to 7, p -values determined by one-way ANOVA.

Article Snippet: MetCCL5 (R&D 335-RM-025) was added to the fresh media at a concentration of 50ng/ml at the beginning of the 24 h shear exposure.

Techniques:

Journal: Oncology Letters

Article Title: Cancer-associated fibroblast-derived CCL5 contributes to cisplatin resistance in A549 NSCLC cells partially through upregulation of lncRNA HOTAIR expression

doi: 10.3892/ol.2021.12957

Figure Lengend Snippet: CAFs secrete elevated CCL5 to promote cell viability in NSCLC cancer cells via paracrine activation. (A) mRNA expression levels of CCL5 in CAFs, NFs and two NSCLC cell lines by RT-qPCR using GAPDH gene as the internal control. (B) CCL5 in conditioned medium secreted by NFs and CAFs were quantified by ELISA. *P<0.05, ***P<0.001 and ****P<0.0001 vs. CAFs. (C) A549 and H1299 cancer cells were incubated with CAF-CM or NF-CM in combination with anti-CCL5 antibody (0.1 µg/ml) for 6 h followed by treatment with DDP for 48 h. Cell viability was determined by the MTT assay. (D) mRNA expression levels of CCL5 and CCR5 in NSCLC cell lines cultured with CAF-CM were assessed by RT-qPCR using GAPDH gene as the normalization control. (E) A549 and H1299 cancer cells were incubated with CAF-CM or NF-CM in combination with CCR5 inhibitor (Met-RANTES; 0.1 µg/ml) for 6 h followed by treatment with DDP for 48 h. Cell viability was determined by the MTT assay. *P<0.05, **P<0.01 and ***P<0.001 vs. control; # P<0.05 and ## P<0.01 vs. DDP; && P<0.01 vs. CAF-CM and DDP. Results were expressed as the mean ± SD, and the means were calculated from ≥3 independent experiments. RT-qPCR, reverse transcription-quantitative PCR; CAF, cancer-associated fibroblast; NF, normal fibroblast; CM, conditioned medium; DDP, cisplatin; CCL5, C-C motif chemokine ligand 5; CCR5, C-C motif chemokine receptor 5; NSCLC, non-small cell lung cancer.

Article Snippet: For cell treatment, cancer cells were incubated with CAF-CM or NF-CM in combination with either anti-CCL5 antibody (0.1 μg/ml; cat. no. MAB678-SP; R&D Systems, Inc.), CCR5 antagonist (Met-RANTES; 0.1 μg/ml; cat. no. 335-RM-025; R&D Systems, Inc.) or recombinant human CCL5 (3 ng/ml; cat. no. 300-06; PeproTech, Inc.) for 6 h, followed by treatment with 50 μM DDP (Sigma-Aldrich; Merck KGaA) in the presence of CM for another 48 h.

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Incubation, MTT Assay, Cell Culture, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Oncology Letters

Article Title: Cancer-associated fibroblast-derived CCL5 contributes to cisplatin resistance in A549 NSCLC cells partially through upregulation of lncRNA HOTAIR expression

doi: 10.3892/ol.2021.12957

Figure Lengend Snippet: CAF-CM increases HOTAIR expression in tumor cells in vitro . (A) HOTAIR expression in human NSCLC A549 and H1299 cell lines treated with CAF-CM or NF-CM was determined by qPCR. HOTAIR expression in human NSCLC A549 and H1299 cell lines treated with CAF-CM and (B) CCL5 neutralizing antibody or (C) CCR5 inhibitor (Met-RANTES) was determined by qPCR. ***P<0.001 and ****P<0.0001 vs. control; ## P<0.01 and ### P<0.001 vs. CAF-CM. Results were expressed as the mean ± SD, and the means were calculated from ≥3 independent experiments. qPCR, quantitative PCR; CAF, cancer-associated fibroblast; NF, normal fibroblast; CM, conditioned medium; CCL5, C-C motif chemokine ligand 5; CCR5, C-C motif chemokine receptor 5; NSCLC, non-small cell lung cancer; HOTAIR, HOX transcript antisense RNA.

Article Snippet: For cell treatment, cancer cells were incubated with CAF-CM or NF-CM in combination with either anti-CCL5 antibody (0.1 μg/ml; cat. no. MAB678-SP; R&D Systems, Inc.), CCR5 antagonist (Met-RANTES; 0.1 μg/ml; cat. no. 335-RM-025; R&D Systems, Inc.) or recombinant human CCL5 (3 ng/ml; cat. no. 300-06; PeproTech, Inc.) for 6 h, followed by treatment with 50 μM DDP (Sigma-Aldrich; Merck KGaA) in the presence of CM for another 48 h.

Techniques: Expressing, In Vitro, Control, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Immunology

Article Title: CCL5 deficiency promotes liver repair by improving inflammation resolution and liver regeneration through M2 macrophage polarization

doi: 10.1038/s41423-019-0279-0

Figure Lengend Snippet: CCL5 induction in DILI patients and an AILI mouse model. a ELISA analysis of serum CCL5 levels in DILI patients (n = 15) and healthy controls (n = 15). b Representative IHC images of hepatic CCL5 expression in DILI patients and healthy controls (original magnification = ×200, scale bar = 100 μm). c Dynamic serum CCL5 levels in WT mice after APAP challenge at the indicated time points (n = 4–6). d Representative IHC images of hepatic CCL5 expression in WT mice 24 h after APAP treatment and in normal controls (original magnification = ×200, scale bar = 100 μm). e Relative Ccl5 mRNA expression was determined in liver tissues, primary hepatocytes (PMHs), and nonparenchymal cells (NPCs) 6 h after APAP treatment (n = 3–4). The data are shown as means ± SEM, *P < 0.05, ****P < 0.0001

Article Snippet: To evaluate the therapeutic potential of CCL5 inhibition, a CCL5-neutralizing antibody (anti-CCL5, AF478, R&D, USA); control IgG (AB-108-C, R&D, USA); or a CCL5 receptor antagonist (Met-CCL5, 335-RM/CF, R&D, USA) were reconstituted in sterile PBS and administered to WT mice (10 μg/injection, i.p) 6 and 24 h after APAP overdose.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing

Journal: Cellular and Molecular Immunology

Article Title: CCL5 deficiency promotes liver repair by improving inflammation resolution and liver regeneration through M2 macrophage polarization

doi: 10.1038/s41423-019-0279-0

Figure Lengend Snippet: Ccl5−/− mice display rapid liver recovery after APAP treatment. a–c APAP treatment in both Ccl5−/− and WT mice at the indicated time points (n = 4–6). a Serum levels of ALT/AST of both genotypes. b Representative images of H&E staining (original magnification = ×100, scale bar = 200 μm) and the statistical quantification of hepatic necrosis. c Representative images of TUNEL staining (original magnification = ×200, scale bar = 100 μm) and the statistical quantification of TUNEL-positive cells. d Survival curve of Ccl5−/− and WT mice in response to a lethal dose of APAP (n = 15). The data are shown as means ± SEM, *P < 0.05, **P < 0.01

Article Snippet: To evaluate the therapeutic potential of CCL5 inhibition, a CCL5-neutralizing antibody (anti-CCL5, AF478, R&D, USA); control IgG (AB-108-C, R&D, USA); or a CCL5 receptor antagonist (Met-CCL5, 335-RM/CF, R&D, USA) were reconstituted in sterile PBS and administered to WT mice (10 μg/injection, i.p) 6 and 24 h after APAP overdose.

Techniques: Staining, TUNEL Assay

Journal: Cellular and Molecular Immunology

Article Title: CCL5 deficiency promotes liver repair by improving inflammation resolution and liver regeneration through M2 macrophage polarization

doi: 10.1038/s41423-019-0279-0

Figure Lengend Snippet: Enhanced inflammation resolution and liver regeneration in Ccl5−/− mice at the late phase of APAP treatment. a–e APAP treatment in both Ccl5−/− and WT mice at the indicated time points (n = 4–6). a ELISA analyses of the serum levels of IL-6 and TNFα. b Representative FACS plots and the statistical quantification of hepatic neutrophils (Ly6G+CD11b+). c Representative IHC images and the statistical quantification of hepatic Ly6G+ cells in liver sections (original magnification = ×400, scale bar = 50 μm). d Representative immunohistochemical staining of hepatic Ki67 in liver sections of Ccl5−/− and control mice at the indicated time points after APAP treatment and the quantification of Ki67-positive cells in liver sections (n = 4–6). e Western blot analysis showing the expression of hepatic PCNA in Ccl5−/− and control mice at the indicated time points after APAP treatment and the quantification of hepatic PCNA levels normalized to β-actin (n = 3–4). The data are shown as means ± SEM, *P < 0.05, **P < 0.01

Article Snippet: To evaluate the therapeutic potential of CCL5 inhibition, a CCL5-neutralizing antibody (anti-CCL5, AF478, R&D, USA); control IgG (AB-108-C, R&D, USA); or a CCL5 receptor antagonist (Met-CCL5, 335-RM/CF, R&D, USA) were reconstituted in sterile PBS and administered to WT mice (10 μg/injection, i.p) 6 and 24 h after APAP overdose.

Techniques: Enzyme-linked Immunosorbent Assay, Immunohistochemical staining, Staining, Western Blot, Expressing

Journal: Cellular and Molecular Immunology

Article Title: CCL5 deficiency promotes liver repair by improving inflammation resolution and liver regeneration through M2 macrophage polarization

doi: 10.1038/s41423-019-0279-0

Figure Lengend Snippet: Increased M2 macrophages in Ccl5−/− mice at the late phase of APAP treatment. a–d APAP treatment in both Ccl5−/− and WT mice at 36 h. a Representative MoMF (CD11bhighF4/80int) and KC (CD11bint F4/80high) profiles of CD45+ liver nongranulocytes from Ccl5−/− and WT mice 36 h after APAP treatment and the quantification of the proportion of MoMFs and KCs relative to total CD45+ liver nongranulocytes (n = 4–6). b Representative FACS plots and the statistical quantification of CD206+ hepatic macrophages (hMφ) (n = 4–6). c The relative expression of M1 markers (iNOS, IL-1β, and TNFα) and M2 markers (Arg1, Ym1, and CD206) was determined in FACS-sorted hepatic macrophages (CD45+CD11b+F4/80+) (n = 4). d Representative images and the statistical quantification of hepatic cells positive for CD68 (a panmacrophage marker) and Ym1 (an M2 macrophage marker) in liver sections (original magnification = ×400, scale bar = 50 μm). The data are shown as the means ± SEM, *P < 0.05, **P < 0.01. hMφ hepatic macrophages

Article Snippet: To evaluate the therapeutic potential of CCL5 inhibition, a CCL5-neutralizing antibody (anti-CCL5, AF478, R&D, USA); control IgG (AB-108-C, R&D, USA); or a CCL5 receptor antagonist (Met-CCL5, 335-RM/CF, R&D, USA) were reconstituted in sterile PBS and administered to WT mice (10 μg/injection, i.p) 6 and 24 h after APAP overdose.

Techniques: Expressing, Marker

Journal: Cellular and Molecular Immunology

Article Title: CCL5 deficiency promotes liver repair by improving inflammation resolution and liver regeneration through M2 macrophage polarization

doi: 10.1038/s41423-019-0279-0

Figure Lengend Snippet: CCL5 directly regulates macrophage polarization. Mouse peritoneal macrophages (PMφ) and bone marrow-derived macrophages (BMMφ) were stimulated with 100 ng/ml rmCCL5 for 6 h, and the expression of M1 macrophage markers (iNOS, IL-1β, and TNFα) was detected by qPCR in PMφ (a) and BMMφ (b). PMφ and BMMφ were stimulated with 20 ng/ml IL4 and with or without 100 ng/ml rmCCL5 for 6 h, and the expression of M2 macrophage markers (Arg1, Ym1, and CD206) was detected by qPCR in PMφ (c) and BMMφ (d). PMφ and BMMφ were stimulated with 20 ng/ml IL4 and with or without 100 ng/ml rmCCL5 for 24 h, and the expression of M2 macrophage markers (Arg1, Ym1, and CD206) was detected by western blot analysis in PMφ (e) and BMMφ (f). The data are represented as the mean ± SEM of at least three independent experiments. **P < 0.01, ***P < 0.001

Article Snippet: To evaluate the therapeutic potential of CCL5 inhibition, a CCL5-neutralizing antibody (anti-CCL5, AF478, R&D, USA); control IgG (AB-108-C, R&D, USA); or a CCL5 receptor antagonist (Met-CCL5, 335-RM/CF, R&D, USA) were reconstituted in sterile PBS and administered to WT mice (10 μg/injection, i.p) 6 and 24 h after APAP overdose.

Techniques: Derivative Assay, Expressing, Western Blot

Journal: Cellular and Molecular Immunology

Article Title: CCL5 deficiency promotes liver repair by improving inflammation resolution and liver regeneration through M2 macrophage polarization

doi: 10.1038/s41423-019-0279-0

Figure Lengend Snippet: CCL5 regulates macrophage polarization mainly through CCR1- and CCR5-related MAPK/NF-κB pathways. a Mouse peritoneal macrophages (PMφ) were stimulated with 100 ng/ml rmCCL5 for 0–60 min, and the signaling activation of the MAPK and NF-κB pathways was detected by western blot analysis. b PMφ were pretreated with a CCR1/3/5 antagonist separately for 1 h and then stimulated with 100 ng/ml rmCCL5 for 30 min. The signaling activation of the MAPK and NF-κB pathways was detected by western blot analysis. c PMφ were pretreated with a CCR1/3/5 antagonist separately for 1 h and then stimulated with 100 ng/ml rmCCL5 for 6 h. qPCR analysis of M1 marker and M2 marker expression was performed (n = 4). d, e Raw 264.7 cells were pretreated with control, CCR1, or CCR5 siRNA separately for 48 h. d Western blot analysis detected the knockdown of CCR1 and CCR5. siRNA-treated cells were stimulated with 100 ng/ml rmCCL5 for 30 min, and the signaling activation of the MAPK and NF-κB pathways was detected by western blot analysis. e siRNA-treated cells were stimulated with 100 ng/ml rmCCL5 for 6 h, and qPCR analysis of M1 marker and M2 marker expression was performed (n = 4). The data are shown as means ± SEM, *P < 0.05. **P < 0.01, ***P < 0.001

Article Snippet: To evaluate the therapeutic potential of CCL5 inhibition, a CCL5-neutralizing antibody (anti-CCL5, AF478, R&D, USA); control IgG (AB-108-C, R&D, USA); or a CCL5 receptor antagonist (Met-CCL5, 335-RM/CF, R&D, USA) were reconstituted in sterile PBS and administered to WT mice (10 μg/injection, i.p) 6 and 24 h after APAP overdose.

Techniques: Activation Assay, Western Blot, Marker, Expressing

Journal: Cellular and Molecular Immunology

Article Title: CCL5 deficiency promotes liver repair by improving inflammation resolution and liver regeneration through M2 macrophage polarization

doi: 10.1038/s41423-019-0279-0

Figure Lengend Snippet: CCL5 neutralization or inhibition facilitates liver recovery after acute liver injury. a A schematic of CCL5 inhibition by anti-CCL5 or Met-CCL5 in the APAP overdose model. The dose of anti-CCL5 and Met-CCL5 was 10 μg. b Serum levels of ALT/AST were detected after anti-CCL5- or Met-CCL5-mediated CCL5 blockage (n = 4–6). c Representative images of H&E staining (original magnification = ×100, scale bar = 200 μm) and the statistical quantification of hepatic necrosis upon anti-CCL5- or Met-CCL5-mediated CCL5 inhibition (n = 4–6). d Representative images and the statistical quantification of hepatic Ly6G+ cells in liver sections (original magnification = ×400, scale bar = 50 μm) upon anti-CCL5- or Met-CCL5-mediated CCL5 inhibition (n = 4–6). e Representative FACS plots and the statistical quantification of CD206+ hepatic macrophages (hMφ) upon anti-CCL5- or Met-CCL5-mediated CCL5 inhibition (n = 4–6). f Survival curves of mice (n = 10–12) in response to a lethal dose of APAP treatment upon anti-CCL5- or Met-CCL5-mediated CCL5 inhibition. The data are shown as means ± SEM, *P < 0.05

Article Snippet: To evaluate the therapeutic potential of CCL5 inhibition, a CCL5-neutralizing antibody (anti-CCL5, AF478, R&D, USA); control IgG (AB-108-C, R&D, USA); or a CCL5 receptor antagonist (Met-CCL5, 335-RM/CF, R&D, USA) were reconstituted in sterile PBS and administered to WT mice (10 μg/injection, i.p) 6 and 24 h after APAP overdose.

Techniques: Neutralization, Inhibition, Staining