Journal: International Journal of Medical Sciences

Article Title: Fat-Storing Multilocular Cells Expressing CCR5 Increase in the Thymus with Advancing Age: Potential Role for CCR5 Ligands on the Differentiation and Migration of Preadipocytes

doi:

Figure Lengend Snippet: CCR5 expression in histological thymic sections of aging mice. Thymus sections of mice with 2, 9, 12 and 18 months of age were submitted to immunoperoxidase using rabbit anti-mouse CCR5 Ab (A-G) or unspecific rabbit IgG (H). Counterstaining was done with hematoxylin. CCR5 was detected in multilocular cells in the septa, parenchyma and adipose tissue (C-G) as well as in thymocytes (F). Black arrows indicate CCR5 + mesenchymal cells in the perithymic adipose tissue (B). White arrows point to fibroblastoid cells originating from the thymic capsule (H). A, adipocyte; Ca, capsule; C, cortex; M, medulla; P, parenchyma; S, septa; mo, months of age. Original magnification: 630x (left) and 1000x (right).

Article Snippet: Primary purified rabbit anti-mouse CCR5 (BD PharMingen, CA), rabbit anti-CCL3, -CCL4 (R&D Systems, MN) or -CCL5 (Chemicon, CA) IgG antibodies were applied on thymic sections at 10μg/ml for 60 min at room temperature in moist humidified chamber.

Techniques: Expressing

Journal: International Journal of Medical Sciences

Article Title: Fat-Storing Multilocular Cells Expressing CCR5 Increase in the Thymus with Advancing Age: Potential Role for CCR5 Ligands on the Differentiation and Migration of Preadipocytes

doi:

Figure Lengend Snippet: CCR5 mRNA expression in the aging thymus. (A) cDNA microarray filters hybridized to total RNA isolated from thymi between 2 and 18 months of age. Arrows indicate spot corresponding to CCR5 mRNA expression. (B) Graphic representation of CCR5 mRNA expression according to cDNA microarray results for thymus of 2, 4, 6, 12 and 18 months of age. (C) CCR5 mRNA expression of thymus of 2, 12 and 18 months old was compared using real time RT/PCR analysis.

Article Snippet: Primary purified rabbit anti-mouse CCR5 (BD PharMingen, CA), rabbit anti-CCL3, -CCL4 (R&D Systems, MN) or -CCL5 (Chemicon, CA) IgG antibodies were applied on thymic sections at 10μg/ml for 60 min at room temperature in moist humidified chamber.

Techniques: Expressing, Microarray, Isolation, Quantitative RT-PCR

Journal: International Journal of Medical Sciences

Article Title: Fat-Storing Multilocular Cells Expressing CCR5 Increase in the Thymus with Advancing Age: Potential Role for CCR5 Ligands on the Differentiation and Migration of Preadipocytes

doi:

Figure Lengend Snippet: Expression of CCR5 and ERTR7 in multilocular mesenchymal cells in the aged thymus . Upper panels show histological sections of 21 month-old thymus containing CCR5-positive cells, by immunoperoxidase. Counterstaining was done with hematoxylin. Upper small panels on the right show CCR5+ multilocular cells inside the thymic parenchyma interacting with thymocytes and stromal cells. Lower panels show multilocular cells stained for ERTR7 (red) and the nuclei dye DAPI (blue), by immunofluorescence. Black arrows indicate CCR5 + multilocular cells. White arrows indicate ERTR7 + multilocular cells. A, adipocyte; C, cortex; S, septae. Original magnification: Upper panels, 1000x; lower panels, 630x.

Article Snippet: Primary purified rabbit anti-mouse CCR5 (BD PharMingen, CA), rabbit anti-CCL3, -CCL4 (R&D Systems, MN) or -CCL5 (Chemicon, CA) IgG antibodies were applied on thymic sections at 10μg/ml for 60 min at room temperature in moist humidified chamber.

Techniques: Expressing, Staining, Immunofluorescence

Journal: International Journal of Medical Sciences

Article Title: Fat-Storing Multilocular Cells Expressing CCR5 Increase in the Thymus with Advancing Age: Potential Role for CCR5 Ligands on the Differentiation and Migration of Preadipocytes

doi:

Figure Lengend Snippet: CCR5 ligands mRNA expression in thymus and thymocytes of young and aged mice. cDNA obtained from thymus (A) or total thymocytes (B) by reverse transcription using RNA isolated from 2- and 18-month-old mice were analyzed by real time RT-PCR. Specific primers to CCL3, CCL4 and CCL5 were utilized. The comparative threshold cycle ( C T ) method was utilized to calculate fold change (mean± SD) between age groups. Student's t test (*p <0.05).

Article Snippet: Primary purified rabbit anti-mouse CCR5 (BD PharMingen, CA), rabbit anti-CCL3, -CCL4 (R&D Systems, MN) or -CCL5 (Chemicon, CA) IgG antibodies were applied on thymic sections at 10μg/ml for 60 min at room temperature in moist humidified chamber.

Techniques: Expressing, Isolation, Quantitative RT-PCR

Journal: International Journal of Medical Sciences

Article Title: Fat-Storing Multilocular Cells Expressing CCR5 Increase in the Thymus with Advancing Age: Potential Role for CCR5 Ligands on the Differentiation and Migration of Preadipocytes

doi:

Figure Lengend Snippet: Fat-storing multilocular cells in the thymus of CCR5-deficient mice. Frozen thymic sections were obtained from 10-11 month-old CCR5-deficient mice (n=3). Histological sections were stained for Oil Red O to identify cells accumulating lipids. Oil Red O + cells were observed in the thymic parenchyma of control and CCR5-deficient mice (green arrows).

Article Snippet: Primary purified rabbit anti-mouse CCR5 (BD PharMingen, CA), rabbit anti-CCL3, -CCL4 (R&D Systems, MN) or -CCL5 (Chemicon, CA) IgG antibodies were applied on thymic sections at 10μg/ml for 60 min at room temperature in moist humidified chamber.

Techniques: Staining

Journal: International Journal of Medical Sciences

Article Title: Fat-Storing Multilocular Cells Expressing CCR5 Increase in the Thymus with Advancing Age: Potential Role for CCR5 Ligands on the Differentiation and Migration of Preadipocytes

doi:

Figure Lengend Snippet: CCR5 ligands promote adipocyte differentiation in vitro . (A) 3T3-L1 cells were fixed and stained, by immunofluorescence, with specific antibodies for mesenchymal cells, ERTR7 (red), and either CCR5 (green), CCL3 (green), CCL4 (green) or CCL5 (green) and the nuclei dye DAPI (blue). Insert shows DAPI and IgG control staining. (B) Confluent 3T3-L1 cells were treated with 100 ng/ml of CCL3, CCL4 or CCL5 for 9 days. After, cell extracts were prepared and analyzed by Western blot using antibodies to the adipocyte differentiation markers PPARγ2 and aP2 as well as to β-actin, as loading control. (C) Increased number of cells differentiated towards adipocyte was observed in confluent 3T3-L1 cells cultured for 9 days (upper panel) and 12 days (lower panels), following treatment in the presence of CCR5 ligands. Chemokines were added every three days in culture media.

Article Snippet: Primary purified rabbit anti-mouse CCR5 (BD PharMingen, CA), rabbit anti-CCL3, -CCL4 (R&D Systems, MN) or -CCL5 (Chemicon, CA) IgG antibodies were applied on thymic sections at 10μg/ml for 60 min at room temperature in moist humidified chamber.

Techniques: In Vitro, Staining, Immunofluorescence, Western Blot, Cell Culture

Journal: International Journal of Medical Sciences

Article Title: Fat-Storing Multilocular Cells Expressing CCR5 Increase in the Thymus with Advancing Age: Potential Role for CCR5 Ligands on the Differentiation and Migration of Preadipocytes

doi:

Figure Lengend Snippet: Migration of 3T3L1 preadipocytes is stimulated by CCR5 ligands in vitro . 3T3-L1 cells were grown to confluence on fibronectin-coated plates (A). The plates were then scratched utilizing a plastic tip (B) and treated or not (C, D) for 20 h with 100 ng/ml of CCL3 (E), CCL4 (F) or CCL5 (G, H). After 20h, control (D) and CCL-5 treated cell cultures (Panel H) were fixed and submitted to Nile Red and DAPI staining. Cells were observed in phase contrast (A-C, E-G) or under fluorescent microscopy (D, H). Migrating cells were observed in the space left by the scratch in the wells. Original magnification: 400x.

Article Snippet: Primary purified rabbit anti-mouse CCR5 (BD PharMingen, CA), rabbit anti-CCL3, -CCL4 (R&D Systems, MN) or -CCL5 (Chemicon, CA) IgG antibodies were applied on thymic sections at 10μg/ml for 60 min at room temperature in moist humidified chamber.

Techniques: Migration, In Vitro, Staining, Microscopy

Journal: Virology

Article Title: HIV entry in macrophages is dependent on intact lipid rafts

doi: 10.1016/j.virol.2008.12.031

Figure Lengend Snippet: HIV receptors are associated with DRMs in macrophages and their levels are altered by MβCD treatment.(A) The surface expression of HIV receptors after MβCD and cholesterol treatment was determined by staining macrophages with fluorescent monoclonal anti-human antibodies to CD4, CCR5, CXCR4, CD71, or an appropriate isotype control. Cell surface binding was measured by flow cytometry, and mean fluorescence intensity values were calculated by subtracting the isotype control fluorescence from the specific antibody fluorescence. Representative FACS plots of macrophages from one donor are shown, and graphs show mean ± SD of data acquired from five different donors. ⁎ Significant difference p < 0.05, ⁎⁎ very significant difference, p < 0.01 and ⁎⁎⁎ extremely significant difference p < 0.001 (paired t -test). (B) Macrophage membranes from lysed cells were incubated in 1% Triton X-100 and then fractionated using an Optiprep step gradient by ultracentrifugation. Fractions were collected, concentrated by TCA precipitation and 20 μL samples of each fraction were analysed by SDS-PAGE using antibodies to CD4, CCR5, flotillin and CD71.

Article Snippet: Protein was transferred onto 0.2 μm PVDF membranes, and membranes were incubated with primary mouse anti-human antibodies to CD4 (clone 34915, 2.5 μg/mL R & D systems) CCR5 (clone CTC5, 2.5 μg/mL R & D systems), flotillin-1 (clone 18, 1.25 μg/mL, BD biosciences) and transferrin receptor (clone 2, 1.25 μg/mL, BD biosciences).

Techniques: Expressing, Staining, Control, Binding Assay, Flow Cytometry, Fluorescence, Incubation, TCA Precipitation, SDS Page

Journal: Virology

Article Title: HIV entry in macrophages is dependent on intact lipid rafts

doi: 10.1016/j.virol.2008.12.031

Figure Lengend Snippet: Nystatin and filipin complex inhibit HIV entry into macrophages. (A) CTB-FITC binding to nystatin and filipin treated cells for 30 min on ice was measured by flow cytometry, with cellular autofluorescence (without CTB-FITC) used as the control. (B) Cell viability after 1 h treatment with nystatin or filipin was determined using the MTS cytotoxicity assay with the number of viable cells being expressed as a % of untreated control cells. (C, D) Productive HIV entry into nystatin (C) or filipin complex (D) treated macrophages was detected after 30 h of infection with HIV BaL by qPCR. (E) HIV-1 infection following nystatin and filipin complex treatment was measured by detecting released virus in supernatant samples by p24 ELISA. (F) Binding of HIV BaL to nystatin (50 μg/mL) and filipin complex (5 μg/mL) treated macrophages for 2 h on ice was measured by p24 ELISA. (G) The surface expression of HIV receptors after nystatin or filipin complex treatment was determined by staining macrophages with fluorescent antibodies to CD4, CCR5, CXCR4, CD71 or an appropriate isotype control. Cell surface binding was measured by flow cytometry, and mean fluorescence intensity values were calculated by subtracting the isotype control fluorescence from the specific antibody fluorescence. Data represent mean ± SD of results obtained with multiple independent experiments (3 donors for qPCR, CTB and p24 binding assays, 4 donors for receptor expression, viability and p24 data is representative of at least 2 donors). ⁎ Significant difference p < 0.05, ⁎⁎ very significant difference, p < 0.01 (paired t -test).

Article Snippet: Protein was transferred onto 0.2 μm PVDF membranes, and membranes were incubated with primary mouse anti-human antibodies to CD4 (clone 34915, 2.5 μg/mL R & D systems) CCR5 (clone CTC5, 2.5 μg/mL R & D systems), flotillin-1 (clone 18, 1.25 μg/mL, BD biosciences) and transferrin receptor (clone 2, 1.25 μg/mL, BD biosciences).

Techniques: Binding Assay, Flow Cytometry, Control, Cytotoxicity Assay, Infection, Virus, Enzyme-linked Immunosorbent Assay, Expressing, Staining, Fluorescence

Journal: Virology

Article Title: HIV entry in macrophages is dependent on intact lipid rafts

doi: 10.1016/j.virol.2008.12.031

Figure Lengend Snippet: Inhibiting cholesterol synthesis inhibits HIV entry into macrophages. (A) Cell viability after 4 days treatment with Lovastatin and/or mevalonate was determined by MTS cytotoxicity assay with the number of viable cells being expressed as a % of untreated control cells. (B) CTB-FITC was bound to Lovastatin-treated macrophages for 30 min at 37 °C and was measured by flow cytometry, with CTB-FITC binding on ice used as the control. (C) Productive HIV BaL entry into macrophages pre-treated with Lovastatin for 4 days was measured after 30 h of infection by qPCR. (D) HIV-1 infection following Lovastatin treatment was measured by detecting released virus in supernatant samples by p24 ELISA. (E) Macrophages were pre-treated with 12.5 μM Lovastatin and/or 800 μg/mL mevalonate (whose production is blocked by Lovastatin) for 4 days. Productive HIV entry was measured by qPCR detection of HIV BaL reverse transcription after 30 h of infection. (F) The surface expression of HIV receptors after Lovastatin treatment was determined by staining macrophages with fluorescent antibodies to CD4, CCR5, CXCR4, CD71 or an appropriate isotype control. Data represent mean ± SD of results obtained with multiple independent experiments (2 donors for CTB and viability assays, 2–5 donors for qPCR, 5–6 donors for receptor expression and p24 data are representative for 2 donors). ⁎⁎⁎ Extremely significant difference p < 0.001 (paired t -test).

Article Snippet: Protein was transferred onto 0.2 μm PVDF membranes, and membranes were incubated with primary mouse anti-human antibodies to CD4 (clone 34915, 2.5 μg/mL R & D systems) CCR5 (clone CTC5, 2.5 μg/mL R & D systems), flotillin-1 (clone 18, 1.25 μg/mL, BD biosciences) and transferrin receptor (clone 2, 1.25 μg/mL, BD biosciences).

Techniques: Cytotoxicity Assay, Control, Flow Cytometry, Binding Assay, Infection, Virus, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Expressing, Staining

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: CCL3–CCR5 axis contributes to progression of esophageal squamous cell carcinoma by promoting cell migration and invasion via Akt and ERK pathways

doi: 10.1038/s41374-020-0441-4

Figure Lengend Snippet: a CCR1 , CCR5 , and CCL3 mRNA expressions in TE-8, TE-9, and TE-15 cells were detected by RT-PCR. The results of western blotting ( b ) and their densitometric analyses ( c , mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.01). CCR1, CCR5, and CCL3 protein expressions in TE-8, TE-9, and TE-15 cells were significantly higher than those of Het-1A cells. However, CCR1 expression levels in TE-8, TE-9, and TE-15 cells were relatively lower than CCL3 and CCR5 expression levels. d CCL3 concentration in conditioned medium of TE-8, TE-9, and TE-15 cells. Protein levels were measured by ELISA. Results are mean ± SEM ( n = 4). CCL3 secretion was detected from TE-8, TE-9, and TE-15 cells. e Immunofluorescence using anti-CCR5 or anti-CCL3 ( green ) antibody on TE-8, TE-9, and TE-15 cells. Nuclei were stained by DAPI ( blue ). CCL3 and CCR5 were expressed in TE-8, TE-9, and TE-15 cells. Magnification: ×400. Scale bar: 10 µm.

Article Snippet: For the IF examination, 1 × 10 5 cultured cells on coverslips were fixed with methanol for 10 min at −20 °C and incubated with primary antibodies against CCR5 (1:25, #sc-32304; Santa Cruz Biotechnology), CCL3 (1:100, #LS-C384561; LifeSpan BioSciences), and CD204 (1:100, #SRA-E5; TransGenic, Kobe, Japan) at 4 °C overnight.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: CCL3–CCR5 axis contributes to progression of esophageal squamous cell carcinoma by promoting cell migration and invasion via Akt and ERK pathways

doi: 10.1038/s41374-020-0441-4

Figure Lengend Snippet: a First, 5 × 10 5 TE-8, TE-9, and TE-15 cells under serum-free condition were treated with 100 ng/ml rhCCL3 for 10, 30, and 60 min. Western blotting was conducted with total protein extracted from the ESCC cell lines using antibodies against total Akt, phosphorylated (p-)Akt (Ser473), p-Akt (Thr308), total ERK, p-ERK (Thr202/Tyr204), and β-actin. Akt and ERK were phosphorylated at 10 min after rhCCL3 treatment in TE-8, TE-9, and TE-15 cells. The results of densitometric analyses were shown in Fig S . b – d First, 5 × 10 5 TE-8, TE-9, and TE-15 cells was transfected by 20 nM siRNA against CCR5 (siCCR5) and negative control siRNA (siNC) for 2 days. The CCR5 knockdown of the ESCC cell lines was confirmed by RT-PCR ( b ) and western blotting using anti-CCR5 ( c ). Western blotting was conducted with total protein extracted from the ESCC cell lines transfected siCCR5 and siNC ( d ). The phosphorylations of Akt and ERK stimulated by rhCCL3 was suppressed by transfection with siCCR5 in TE-8, TE-9, and TE-15 cells. The results of densitometric analyses were shown in Fig S .

Article Snippet: For the IF examination, 1 × 10 5 cultured cells on coverslips were fixed with methanol for 10 min at −20 °C and incubated with primary antibodies against CCR5 (1:25, #sc-32304; Santa Cruz Biotechnology), CCL3 (1:100, #LS-C384561; LifeSpan BioSciences), and CD204 (1:100, #SRA-E5; TransGenic, Kobe, Japan) at 4 °C overnight.

Techniques: Western Blot, Transfection, Negative Control, Knockdown, Reverse Transcription Polymerase Chain Reaction

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: CCL3–CCR5 axis contributes to progression of esophageal squamous cell carcinoma by promoting cell migration and invasion via Akt and ERK pathways

doi: 10.1038/s41374-020-0441-4

Figure Lengend Snippet: a Representative CCL3 immunoreactivities in human ESCC tissues. The immunoreactivity in the tumor nests was assessed and divided into low ( n = 20) and high ( n = 48) groups based on the staining intensity. Typical images are shown: low expression (i, iii) and high expression (ii, iv). Areas delimited by squares in (i) and (ii) (magnification: ×100; scale bar: 100 µm) are showed in higher magnification in (iii) and (iv) (magnification: ×400; scale bar: 20 µm), respectively. b Representative CCR5 immunoreactivities in human ESCC tissues. Immunoreactivity in the tumor nest was assessed and divided into low ( n = 25) and high ( n = 43) groups based on the staining intensity. Typical images are shown: low expression (i, iii) and high expression (ii, iv). Areas delimited by squares in (i) and (ii) (magnification: ×100; scale bar: 100 µm) are showed in higher magnification in (iii) and (iv) (magnification: ×400; Scale bar: 20 µm), respectively. c Double immunofluorescence was performed using antibodies against CCL3 ( green ) and CD204 ( red ). CCL3 was expressed in both cancer cells and CD204 + TAMs. Nuclei were stained by DAPI ( blue ). White dotted line indicates the edge of the tumor nest. Magnification: ×400. Scale bar: 10 µm.

Article Snippet: For the IF examination, 1 × 10 5 cultured cells on coverslips were fixed with methanol for 10 min at −20 °C and incubated with primary antibodies against CCR5 (1:25, #sc-32304; Santa Cruz Biotechnology), CCL3 (1:100, #LS-C384561; LifeSpan BioSciences), and CD204 (1:100, #SRA-E5; TransGenic, Kobe, Japan) at 4 °C overnight.

Techniques: Staining, Expressing, Immunofluorescence

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: CCL3–CCR5 axis contributes to progression of esophageal squamous cell carcinoma by promoting cell migration and invasion via Akt and ERK pathways

doi: 10.1038/s41374-020-0441-4

Figure Lengend Snippet: Correlations between CCL3 and/or CCR5 expression and clinicopathological factors.

Article Snippet: For the IF examination, 1 × 10 5 cultured cells on coverslips were fixed with methanol for 10 min at −20 °C and incubated with primary antibodies against CCR5 (1:25, #sc-32304; Santa Cruz Biotechnology), CCL3 (1:100, #LS-C384561; LifeSpan BioSciences), and CD204 (1:100, #SRA-E5; TransGenic, Kobe, Japan) at 4 °C overnight.

Techniques: Expressing

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: CCL3–CCR5 axis contributes to progression of esophageal squamous cell carcinoma by promoting cell migration and invasion via Akt and ERK pathways

doi: 10.1038/s41374-020-0441-4

Figure Lengend Snippet: The patients were divided into low and high CCL3 expression groups ( a ); low and high CCR5 expression groups ( b ); high expression of both CCL3 and CCR5 (H/H) and all the other patients (non-H/H) groups ( c ). The data were analyzed by log-rank test (* p < 0.05; ** p < 0.01; ns not significant).

Article Snippet: For the IF examination, 1 × 10 5 cultured cells on coverslips were fixed with methanol for 10 min at −20 °C and incubated with primary antibodies against CCR5 (1:25, #sc-32304; Santa Cruz Biotechnology), CCL3 (1:100, #LS-C384561; LifeSpan BioSciences), and CD204 (1:100, #SRA-E5; TransGenic, Kobe, Japan) at 4 °C overnight.

Techniques: Expressing

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: CCL3–CCR5 axis contributes to progression of esophageal squamous cell carcinoma by promoting cell migration and invasion via Akt and ERK pathways

doi: 10.1038/s41374-020-0441-4

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis of prognostic factors for disease-free survival.

Article Snippet: For the IF examination, 1 × 10 5 cultured cells on coverslips were fixed with methanol for 10 min at −20 °C and incubated with primary antibodies against CCR5 (1:25, #sc-32304; Santa Cruz Biotechnology), CCL3 (1:100, #LS-C384561; LifeSpan BioSciences), and CD204 (1:100, #SRA-E5; TransGenic, Kobe, Japan) at 4 °C overnight.

Techniques: Expressing

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: CCL3–CCR5 axis contributes to progression of esophageal squamous cell carcinoma by promoting cell migration and invasion via Akt and ERK pathways

doi: 10.1038/s41374-020-0441-4

Figure Lengend Snippet: CCL3 is derived from both TAMs and cancer cells and is bound to CCR5 on cancer cells. CCL3–CCR5 interaction contributes to the progression of ESCC by activating Akt and ERK signaling pathways and by promoting the migration and invasion of cancer cells and angiogenesis.

Article Snippet: For the IF examination, 1 × 10 5 cultured cells on coverslips were fixed with methanol for 10 min at −20 °C and incubated with primary antibodies against CCR5 (1:25, #sc-32304; Santa Cruz Biotechnology), CCL3 (1:100, #LS-C384561; LifeSpan BioSciences), and CD204 (1:100, #SRA-E5; TransGenic, Kobe, Japan) at 4 °C overnight.

Techniques: Derivative Assay, Protein-Protein interactions, Migration

Journal: BioMed Research International

Article Title: MiR-455-5p Suppresses the Progression of Prostate Cancer by Targeting CCR5

doi: 10.1155/2019/6394784

Figure Lengend Snippet: CCR5 as a direct target of miR-455-5p. (a) Diagram of putative miR-455 binding sites in the 3′-UTR (397–412) of CCR5 mRNA. (b) Relative activities of luciferase reporters containing CCR5 3′-UTR variants cotransfected with miR-455-5p or negative-control mimics in LNCAP and DU145 cells. (c) Protein levels of CCR5 72 h after miR-455-5p and inhibitor transfection in LNCAP and DU145 cells. ∗ , P < 0.05; ∗∗ , P < 0.01; ∗∗∗ , P < 0.001.

Article Snippet: The paraffin-embedded xenograft tumors for IHC were processed in accordance with the manufacturer's instructions using a primary antibody: CCR5 mix (1:250; Protein Tech, Wuhan, China).

Techniques: Binding Assay, Luciferase, Negative Control, Transfection

Journal: BioMed Research International

Article Title: MiR-455-5p Suppresses the Progression of Prostate Cancer by Targeting CCR5

doi: 10.1155/2019/6394784

Figure Lengend Snippet: Regulation of cell proliferation and apoptosis by miR-455-5p through directly targeting CCR5. (a) Protein levels of CCR5 in LNCAP and DU145 cells 72 h after transfection. (b–d) Cell proliferation was assessed by CCK-8 and colony-formation assays in LNCAP and DU145 cells after cotransfection. (e) Cell apoptosis was analyzed by annexin V-FITC plus PI staining in LNCAP and DU145 cells. (f) Western blot revealed the protein levels of caspase 3 and cleaved caspase 3 and CCR5 in LNCAP and DU145 cells 72 h after cotransfection. (g) Representative images of IHC for CCR5 on xenografts. Brown signals on the cell membrane represented CCR5 expression level (Magnification, ×200 bar). (h) Inverse correlation of CCR5 and miR-455-5p in the GSE20132 database (r = −0.28, P = 0.0025). (i) Kaplan–Meier curves for the RFS of patients with PCa stratified by tissue CCR5 levels. ∗ , P < 0.05.

Article Snippet: The paraffin-embedded xenograft tumors for IHC were processed in accordance with the manufacturer's instructions using a primary antibody: CCR5 mix (1:250; Protein Tech, Wuhan, China).

Techniques: Transfection, CCK-8 Assay, Cotransfection, Staining, Western Blot, Membrane, Expressing

Journal: Annals of Dermatology

Article Title: The Contributory Roles of Th17 Lymphocyte and Cytotoxic T Lymphocyte at the Hair Bulge Region as Well as the Hair Bulb Area in the Chronic Alopecia Areata Patients

doi: 10.5021/ad.2017.29.2.156

Figure Lengend Snippet: Expression of CCR6 and CCR5 within type 1 hair bulbs (B~D), type 2 hair bulbs (F~H), and type 3 hair bulbs (J~L). A representative finding of double immunofluorescences immunoreactive cells for CCR6 (B, F, J; green) and CCR5 (C, G, K; red) with colocalization study of CCR6 and CCR5 (D, H, L). H&E stained sectionswere also shown from type 1 to type 3 (A, E, I). The denser CCR6 and CCR5 were expressed in parallel with the severer histopathologic gradings (H&E: A, E, I, ×100; double immunofluorescence, B~D, F~H, J~L, ×100).

Article Snippet: The following primary antibodies were used: mouse monoclonal anti-CCR5 antibody (clone 45549; R&D Systems, Abingdon, UK) for the Th1 fraction, and rabbit polyclonal anti-CCR6 antibody (clone ab78429; Abcam, Cambridge, UK) for the Th17 fraction.

Techniques: Expressing, Staining, Immunofluorescence

Journal: Arhiv za bioloske nauke

Article Title: The role of ccr5 polymorphism in colorectal cancer and liver metastasis in the Tunisian population

doi: 10.2298/abs210817044w

Figure Lengend Snippet: Fig. 2. Immunohistochemical analysis of CCR5-staining cells in CRC and CRLM tissues (scale bar=10 μm). Cytoplasmic moderate to weak positivity with anti-CCR5 in the glandular structures and the lamina propria of healthy colorectal tissue (A); intense posi- tivity in healthy liver tissue (B); intense membrane-cytoplasmic staining of cancerous cells with an attenuated signal at the stroma in well-differentiated CRC (C); moderate cytoplasmic positivity in moderately differentiated CRC (D); weak to absent tumor cell staining, with vascular basal membrane positivity in poorly dif- ferentiated CRC (E); exclusive cytoplasmic staining for the (Δ32/ Δ32) homozygous CRC patients (F); discontinuous membrane staining for the heterozygous patients (wt/Δ32) with moderate to intense cytoplasmic CCR5 expression (G); intense membranous and stromal positive staining with anti-CCR5 in well-differenti- ated CRLM (H); moderate cytoplasmic positivity in moderately differentiated CRLM (I); moderate to poor positivity in poorly differentiated CRLM (J); heterogeneous and discontinuous posi- tivity of glandular structures, with negativity in the mucus and positivity in the fibroinflammatory stroma with anti- CCR5 in mucinous carcinoma (K). A and B magnification ×100; C, F and H-K magnification ×250; D, E and G magnification ×400.

Article Snippet: All the samples were then exposed to peroxidase block (Novolink Polymer Detection Kit; Leica Biosystems, Newcastle, UK) for 5 min, then incubated overnight at 4°C with primary antibody to CCR5 (1:50, Monoclonal Mouse IgG2B clone, R&D Systems, Minneapolis, MN, USA).

Techniques: Immunohistochemical staining, Staining, Membrane, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Histidyl–tRNA Synthetase and Asparaginyl–tRNA Synthetase, Autoantigens in Myositis, Activate Chemokine Receptors on T Lymphocytes and Immature Dendritic Cells

doi: 10.1084/jem.20020186

Figure Lengend Snippet: (A) CCR5 transfected HEK/293 cells (CCR5/HEK) migrate to HisRS. (B) CCR3 transfected HEK/293 cells (CCR3/HEK) migrate to AsnRS and SerRS. Recombinant HisRS, SerRS, or AsnRS was placed in the lower wells of a micro-Boyden chamber. The open circles (CCR3) and open squares (CCR1) show the lack of chemotaxis of CCR3 and CCR1 transfected HEK cells to HisRS. The concentrations of are shown on the x-axis while the mean number of cells in a 200× filed ± standard deviation is shown on the y-axis. Each condition was performed in triplicate. Each assay was performed a minimum of three times. Unpaired Student t tests were performed comparing the number of migrating cells in the binding media vs. the number of migrating cells in individual chemoattractant concentrations. * denotes a P value ≤0.0001.

Article Snippet: The sections were incubated overnight at 4°C with a 1:50 dilution of primary monoclonal anti–human CCR5 or anti–human CCR3 (R&D Systems) and with anti–mouse horseradish peroxidase (HRP; Dako) for 1 h at room temperature.

Techniques: Transfection, Recombinant, Chemotaxis Assay, Standard Deviation, Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: Histidyl–tRNA Synthetase and Asparaginyl–tRNA Synthetase, Autoantigens in Myositis, Activate Chemokine Receptors on T Lymphocytes and Immature Dendritic Cells

doi: 10.1084/jem.20020186

Figure Lengend Snippet: Migration of CCR5 and CCR2 chimeras to HisRS or 1–48 HRS. CCR5, CCR2, and chimeric constructs were expressed in HEK/293 cells. Recombinant HisRS (HRS) or the 1–48 HRS peptide was placed in the lower wells of a micro-Boyden chamber. The concentrations of HRS is shown on the x-axis while the mean number of cells in a 200× field ± standard deviation is shown on the y-axis. Each condition was performed in triplicate. Each assay was performed a minimum of three times. Unpaired Student t tests were performed comparing the number of migrating cells in the binding media vs. the number of migrating cells in individual chemoattractant concentrations. * denotes a P value ≤0.0001, ** denotes a P value of ≤0.001.

Article Snippet: The sections were incubated overnight at 4°C with a 1:50 dilution of primary monoclonal anti–human CCR5 or anti–human CCR3 (R&D Systems) and with anti–mouse horseradish peroxidase (HRP; Dako) for 1 h at room temperature.

Techniques: Migration, Construct, Recombinant, Standard Deviation, Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: Histidyl–tRNA Synthetase and Asparaginyl–tRNA Synthetase, Autoantigens in Myositis, Activate Chemokine Receptors on T Lymphocytes and Immature Dendritic Cells

doi: 10.1084/jem.20020186

Figure Lengend Snippet: (A) CCR5 neutralizing antibody blocks CCL4, CCL5, and HisRS-induced CCR5/HEK migration. (B) CCL5 and HisRS (HRS) block domain specific anti-CCR5 binding. CCR5/HEK cells were pretreated with either isotype control antibody, shown in hatched bars, or neutralizing antibody 45531, shown in solid bars, then subjected to chemotaxis. Pretreatment with 45531 significantly inhibited all induced chemotaxis (* indicates P < 0.0001 between sets designated by a bracket), but did not inhibit spontaneous migration (*** indicates P > 0.2). A representative FACS ® experiment of three is shown. Pretreatment with CCL5 blocked multidomain specific monoclonal antibody 45523 binding to a greater extend than did HisRS (HRS). However, both CCL5 and HisRS blocked extracellular loop 2 specific antibody 45531 binding, equally.

Article Snippet: The sections were incubated overnight at 4°C with a 1:50 dilution of primary monoclonal anti–human CCR5 or anti–human CCR3 (R&D Systems) and with anti–mouse horseradish peroxidase (HRP; Dako) for 1 h at room temperature.

Techniques: Migration, Blocking Assay, Binding Assay, Control, Chemotaxis Assay

Journal: The Journal of Experimental Medicine

Article Title: Histidyl–tRNA Synthetase and Asparaginyl–tRNA Synthetase, Autoantigens in Myositis, Activate Chemokine Receptors on T Lymphocytes and Immature Dendritic Cells

doi: 10.1084/jem.20020186

Figure Lengend Snippet: Immunohistochem-istry for CCR5 and CCR3. Muscle biopsy slides from patients with myositis were stained with anti–human CCR5 (A) or anti–human CCR3 (B). Arrows point to several CCR5 and CCR3-positive mononuclear cells. Isotype controls did not stain any infiltrating mononuclear cells (C). Original magnification: 340×.

Article Snippet: The sections were incubated overnight at 4°C with a 1:50 dilution of primary monoclonal anti–human CCR5 or anti–human CCR3 (R&D Systems) and with anti–mouse horseradish peroxidase (HRP; Dako) for 1 h at room temperature.

Techniques: Staining

Journal: The Journal of Experimental Medicine

Article Title: Histidyl–tRNA Synthetase and Asparaginyl–tRNA Synthetase, Autoantigens in Myositis, Activate Chemokine Receptors on T Lymphocytes and Immature Dendritic Cells

doi: 10.1084/jem.20020186

Figure Lengend Snippet: Myositis patients have fewer CD14 + CCR5 + circulating leukocytes. Peripheral blood was collected from unaffected individuals ( n = 8) and anti-Jo-1–positive myositis patients ( n = 7). Peripheral mononuclear cells were collected, stained for CD3, CD14, CCR5, or CXCR4 and subjected to FACS ® analysis. Median values with standard deviations are plotted. The reduction observed between unaffected samples and affected samples is statistically significant as determined by paired Student t test P = 0.036.

Article Snippet: The sections were incubated overnight at 4°C with a 1:50 dilution of primary monoclonal anti–human CCR5 or anti–human CCR3 (R&D Systems) and with anti–mouse horseradish peroxidase (HRP; Dako) for 1 h at room temperature.

Techniques: Staining

Journal: Antiviral research

Article Title: Binding of fusion protein FLSC IgG1 to CCR5 is enhanced by CCR5 antagonist Maraviroc

doi: 10.1016/j.antiviral.2014.10.006

Figure Lengend Snippet: Competition between FLSC IgG1 and MAbs for CCR5. JC53 cells were stained with FLSC IgG1 alone or in competition with CCR5 mAbs. Panel A shows cells treated with FLSC IgG1 Zenon Alexa 488, Panel B shows cells treated with ECL2 mAb and FLSC IgG1 Zenon Alexa 488, Panel C shows cells treated with the N-terminus antibody and FLSC IgG1 Zenon Alexa 488, and Panel D shows cells after combined and simultaneous treatment with both mAbs and FLSC IgG1 Zenon Alexa 488. Panel E shows the normalized relative staining intensity of CCR5 in samples pretreated or not with antibodies and then with FLSC IgG1 Zenon Alexa 488. AF samples were an unstained autofluorescence control. Bar size is 10 µm.

Article Snippet: Cells were washed again, corresponding secondary antibody was added, and cells were incubated on ice for 90 min. 2D7 primary antibody (purified mouse antihuman CCR5 mAb, clone 2D7/CCR5, BD Biosciences) stained with PE rat anti-mouse IgG2a + b mAb (clone X57, BD Biosciences) secondary antibody.

Techniques: Staining

Journal: Antiviral research

Article Title: Binding of fusion protein FLSC IgG1 to CCR5 is enhanced by CCR5 antagonist Maraviroc

doi: 10.1016/j.antiviral.2014.10.006

Figure Lengend Snippet: Contacts of FLSC IgG1 and CCR5. FLSC IgG1 Zenon Alexa 488 (green – Panel A) binding to CCR5 stained with CCR5 mAb, clone 45523 and an Alexa 647 conjugated secondary antibody (red – Panel B), their overlap (yellow – Panel C) and a 3-D close up of direct contacts between CCR5 and FLSC IgG1 on the TZMbl cell membrane (Panels D and E) were imaged by super resolution microscopy (STORM) and total internal reflection fluorescence methodology (TIRF) (Rust et al., 2006).

Article Snippet: Cells were washed again, corresponding secondary antibody was added, and cells were incubated on ice for 90 min. 2D7 primary antibody (purified mouse antihuman CCR5 mAb, clone 2D7/CCR5, BD Biosciences) stained with PE rat anti-mouse IgG2a + b mAb (clone X57, BD Biosciences) secondary antibody.

Techniques: Binding Assay, Staining, Microscopy, Fluorescence

Journal: Antiviral research

Article Title: Binding of fusion protein FLSC IgG1 to CCR5 is enhanced by CCR5 antagonist Maraviroc

doi: 10.1016/j.antiviral.2014.10.006

Figure Lengend Snippet: Entry inhibition by FLSC compared with FLSC IgG1. Shown are comparisons of HIV-1 entry inhibition by FLSC and FLSC IgG1 in high CCR5-expressing JC53 cells (Panel A), low CCR5-expressing JC57 cells (Panel B), and primary human PBMCs (Panel C). Results were from BlaM fusion assays.

Article Snippet: Cells were washed again, corresponding secondary antibody was added, and cells were incubated on ice for 90 min. 2D7 primary antibody (purified mouse antihuman CCR5 mAb, clone 2D7/CCR5, BD Biosciences) stained with PE rat anti-mouse IgG2a + b mAb (clone X57, BD Biosciences) secondary antibody.

Techniques: Inhibition, Expressing

Journal: Antiviral research

Article Title: Binding of fusion protein FLSC IgG1 to CCR5 is enhanced by CCR5 antagonist Maraviroc

doi: 10.1016/j.antiviral.2014.10.006

Figure Lengend Snippet: Antiviral activities of FLSC IgG1 and CCR5 antibodies. Antiviral activity was measured by single cycle pseudovirus infection and quantified by luciferase activity. Solid circles represent the antiviral activity of FLSC IgG1; open squares, 2D7; triangles, CD195; open circles, anti-CCR5 NT; and solid diamonds, 45523. Experiments were done twice using JC57 cells and each result done in triplicate. Data are given ±SD.

Article Snippet: Cells were washed again, corresponding secondary antibody was added, and cells were incubated on ice for 90 min. 2D7 primary antibody (purified mouse antihuman CCR5 mAb, clone 2D7/CCR5, BD Biosciences) stained with PE rat anti-mouse IgG2a + b mAb (clone X57, BD Biosciences) secondary antibody.

Techniques: Activity Assay, Infection, Luciferase

Journal: Antiviral research

Article Title: Binding of fusion protein FLSC IgG1 to CCR5 is enhanced by CCR5 antagonist Maraviroc

doi: 10.1016/j.antiviral.2014.10.006

Figure Lengend Snippet: Effect of CCR5 densities on antiviral activity by FLSC IgG1. Panel A shows the effect of CCR5 density on antiviral activity of FLSC IgG1 in cell lines. JC10 cells (2000 CCR5 molecules/cell) and JC57 cells (9000 CCR5 molecules/cell) were infected with replication competent R5 Bal HIV-1 in the presence of the indicated concentrations of FLSC IgG1. Panel B shows that CCR5 density on primary CD4+ T cells correlates inversely with the antiviral activity of FLSC IgG1. Activated PBMCs from different donors were subjected to flow cytometry analysis, and then infected with Bal HIV-1 in the presence of varying concentrations of FLSC IgG1. Panel C shows effect of CCR5 density using a different R5 HIV-1 isolate (CC 1/85) to infect JC10 and JC57 cells. Virus production was measured by p24 ELISA on day 4 or 5 after infection and the data used to determine EC50 values. Data are presented as averages of triplicates ± SD from two experiments. FLSC IgG1 EC50 values were plotted against CCR5 density values (listed in the legend) on the day of infection. Line fit was done by linear regression where r is Spearman correlation coefficient (r = 0.90 – red line).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Cells were washed again, corresponding secondary antibody was added, and cells were incubated on ice for 90 min. 2D7 primary antibody (purified mouse antihuman CCR5 mAb, clone 2D7/CCR5, BD Biosciences) stained with PE rat anti-mouse IgG2a + b mAb (clone X57, BD Biosciences) secondary antibody.

Techniques: Activity Assay, Infection, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Antiviral research

Article Title: Binding of fusion protein FLSC IgG1 to CCR5 is enhanced by CCR5 antagonist Maraviroc

doi: 10.1016/j.antiviral.2014.10.006

Figure Lengend Snippet: CCR5 binding by FLSC IgG1, MVC and FLSCIgG1 plus MVC. Data present the relative fluorescence intensity of residual CCR5 on JC57 cells (9000 CCR5 molecules/cell) stained with CCR5 antibody clone 45531 plus biotin and streptavidin amplification of signal, upon treatment with FLSC IgG1 alone, MVC alone and combined treatment. The isotype control shows minimal fluorescence, as expected. The data are presented as averages of triplicates with ±SD from three independent experiments. Data are shown as percentages and normalized to CCR5 intensity values obtained in the absence of FLSC IgG1 or MVC.

Article Snippet: Cells were washed again, corresponding secondary antibody was added, and cells were incubated on ice for 90 min. 2D7 primary antibody (purified mouse antihuman CCR5 mAb, clone 2D7/CCR5, BD Biosciences) stained with PE rat anti-mouse IgG2a + b mAb (clone X57, BD Biosciences) secondary antibody.

Techniques: Binding Assay, Fluorescence, Staining, Amplification

Journal: Antiviral research

Article Title: Binding of fusion protein FLSC IgG1 to CCR5 is enhanced by CCR5 antagonist Maraviroc

doi: 10.1016/j.antiviral.2014.10.006

Figure Lengend Snippet: Measurements of bound CCR5 molecules with different anti-CCR5 mAb with or without 10 lM MVC treatment on JC53 cell line using plasmonic substrate methodology. Panel A shows average intensities of cells CCR5 molecules expressed on JC53 cells stained with FLSC IgG1 Alexa 555. The color coded symbols present numbered cells from three different images (three colors). Mean intensities (dashed line) and SD were calculated based on multiple cells within three images and corrected for cell autofluorescence (unstained cells). Number of cells varied within the image from 15 to 20 and were numbered from 1 to 20 within image for MVC(—) and from 25 to 25 for MVC(+). The p value was calculated based on mean, standard deviation and number of cells. Panel B shows normalized mean intensities of JC53 cells untreated or treated with MVC and imaged using different sets of CCR5 primary (2D7, HGS101, and FLSC IgG1) and dye-labeled secondary antibodies. The change in CCR5 expression is proportional to change in intensity. Presented data are of two independent experiments in triplicates, three cell images, with ±SD. The p values were calculated similarly as described in Panel A, (*) mean p < 0.0001.

Article Snippet: Cells were washed again, corresponding secondary antibody was added, and cells were incubated on ice for 90 min. 2D7 primary antibody (purified mouse antihuman CCR5 mAb, clone 2D7/CCR5, BD Biosciences) stained with PE rat anti-mouse IgG2a + b mAb (clone X57, BD Biosciences) secondary antibody.

Techniques: Staining, Standard Deviation, Labeling, Expressing