Journal: Frontiers in Immunology

Article Title: CD4 + T Follicular Helper and IgA + B Cell Numbers in Gut Biopsies from HIV-Infected Subjects on Antiretroviral Therapy Are Similar to HIV-Uninfected Individuals

doi: 10.3389/fimmu.2016.00438

Figure Lengend Snippet: (A) Indirect immunofluorescence to measure CCR5 + CD4 + T cells. Representative flow plot on left shows negative control where anti-CCR5 primary monoclonal antibody was omitted. Flow plot on right shows CCR5 expression on memory (CD45RA − ) CD4 + T cells from a TI biopsy from a healthy control (HNC). (B) CCR5 + % of memory CD4 + and CCR5 + % of memory CD45RO + CD8 + T cells in gut biopsies from HIV-negative controls (HNC) compared with HIV + subjects on ART, respectively, for left colon (LC) and terminal ileum (TI). (C) Representative flow plot on right shows co-expression of CD38 and HLA-DR on CCR5 + memory CD4 + T cells (gated on left, from (A) , above) from a TI biopsy from a healthy control (HNC). (D) Co-expression of CD38 and HLA-DR on CCR5 + memory CD4 + and CCR5 + memory CD8 + T cells, in gut biopsies from HIV-negative controls (HNC) compared with HIV + subjects on ART, respectively, for left colon (LC) and terminal ileum (TI).

Article Snippet: Gut biopsy cells were stained with 10 μg/ml purified mAb against CCR5 (clone 2D7; BD Biosciences) for 15 min at RT, washed once with PBA, and incubated for 15 min at RT with a 1:50 dilution of PE-conjugated F(ab′) 2 fragment goat anti-mouse IgG (H + L chain-specific) PE (human, bovine, rabbit, swine serum protein absorbed) secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

Techniques: Immunofluorescence, Negative Control, Expressing

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: Journal of Virology

Article Title: cis Expression of DC-SIGN Allows for More Efficient Entry of Human and Simian Immunodeficiency Viruses via CD4 and a Coreceptor

doi: 10.1128/jvi.75.24.12028-12038.2001

Figure Lengend Snippet: FIG. 1. cis expression of DC-SIGN enhances viral infection. (A) One microgram each of plasmids expressing CD4, coreceptor (CCR5 or CXCR4), and DC-SIGN or vector alone (pcDNA3) was transfected into 293T cells in each well of a 24-well plate. Twenty hours postinfection, the transfected cells were infected with the indicated pseudotyped luciferase reporter viruses. Cells were lysed 4 days postinfection, and luciferase activity was detected as described in Materials and Methods. Results are not shown for ADA and SIV pseudotypes on CXCR4-transfected cells and IIIB pseudotypes on CCR5-transfected cells because they did not result in reproducible infections above the background in the presence or absence of DC-SIGN. Results are representative of three experiments performed in duplicates or triplicates. (B) Infections were performed as in panel A, except that target cells were transfected with the equivalent of 20 ng of CCR5 expression plasmid where indicated. Shown here are averages from infections performed in duplicates. Average raw relative light units are indicated above each bar, so that results from panels A and B can be compared directly.

Article Snippet: Samples were then blotted for CD4 with rabbit anti-CD4 polyclonal antibodies (previously generated by immunizing New Zealand White rabbits with the recombinant soluble 4 domain, CD4) or a mouse MAb against CCR5 (CTC5; R&D Systems, Minneapolis, Minn.).

Techniques: Expressing, Infection, Plasmid Preparation, Transfection, Luciferase, Activity Assay

Journal: Journal of Virology

Article Title: cis Expression of DC-SIGN Allows for More Efficient Entry of Human and Simian Immunodeficiency Viruses via CD4 and a Coreceptor

doi: 10.1128/jvi.75.24.12028-12038.2001

Figure Lengend Snippet: FIG. 3. DC-SIGN can enhance viral entry via limiting levels of CCR5. (A) A total of 105 DC-SIGN-transduced Jurkat or SupT1 cells were infected with 0.2 ng of ADA as in Fig. 2. p24 antigen levels in the culture supernatant were determined at days 1, 4, and 8 postinfection. (B) DC-SIGN-transduced Jurkat cells were infected with two different strains of R5 viruses (2 ng each of ADA and YU2) in the absence (UnRx) or presence of anti-CXCR4 (20 g of MAb 45701 per ml), anti-CD4 (10 g of Leu3A per ml), or TAK779 (20 M). Culture supernatants were half-exchanged on days 3, 6, and 9 with fresh growth media and the appropriate blocking agents as indicated. p24 antigen levels were determined with a commercial ELISA kit. The results shown are averages of experiments done in triplicate. (C) Retrovirally (MIGR1–DC-SIGN–GFP) transduced SupT1 and Jurkat cells were stained for DC-SIGN with DC028, and the number of antibody binding sites was determined by QFACS analysis with the Quantum Simply Cellular kit (Sigma). Note that the MIGR1 vector expresses DC-SIGN in tandem with GFP via an IRES linker. (D) cis enhancement effect on DC-SIGN-transduced Jurkat cells can be inhibited by mannan. HxB or SIV316 psuedotyped GFP reporter viruses were used to infect parental Jurkat or DC-SIGN-transduced Jurkat cells in the presence of absence of 100 g of mannan per ml as described in Materials and Methods. Productive infection was determined by staining for intracellular p24 antigen 3 days postinfection. Results are presented as fold enhancement: that is, the percentage of p24 cells obtained with Jurkat-DC-SIGN cells divided by the percentage of p24 cells obtained with Jurkat parental cells. For comparison, the fold enhancement obtained with parental Jurkat cells is shown and is, by definition, normalized to a value of 1.

Article Snippet: Samples were then blotted for CD4 with rabbit anti-CD4 polyclonal antibodies (previously generated by immunizing New Zealand White rabbits with the recombinant soluble 4 domain, CD4) or a mouse MAb against CCR5 (CTC5; R&D Systems, Minneapolis, Minn.).

Techniques: Infection, Blocking Assay, Enzyme-linked Immunosorbent Assay, Staining, Binding Assay, Plasmid Preparation, Comparison

Journal: Journal of Virology

Article Title: cis Expression of DC-SIGN Allows for More Efficient Entry of Human and Simian Immunodeficiency Viruses via CD4 and a Coreceptor

doi: 10.1128/jvi.75.24.12028-12038.2001

Figure Lengend Snippet: FIG. 6. DC-SIGN expression on permissive tissue macrophages. Immunohistochemistry was performed on tissue sections as indicated. Staining for CD14 (macrophage marker) and DC-SIGN was performed on 5-m serial sections in order to show costaining of CD14 and DC-SIGN on the same cell. (A) (Lymph node) CD14 macrophages (arrows in left panel) in the germinal centers appear negative for DC-SIGN. (DC-SIGN expression is indicated by brown cells in the right panel.) (Spleen) Numerous CD14 macrophages are scattered throughout the red and white pulp (left panel); the few DC-SIGN-positive cells (right panel, arrows) appear CD14. (Alveoli) DC-SIGN-positive cells in the alveoli also express low levels of CD14 (arrows in right and left panels point to cells staining for both markers). Alveolar macrophages are a heterogeneous population and can vary in their amount of CD14 expression. DC-SIGN-positive cells in the alveoli appear to be restricted to CD14low cells (arrows). (B) Confocal microscopy performed on alveolar macrophages showing expression of DC-SIGN (red) with CCR5 (green) and CD4 (green). Note the almost complete colocalization of DC-SIGN with CCR5 (yellow overlap).

Article Snippet: Samples were then blotted for CD4 with rabbit anti-CD4 polyclonal antibodies (previously generated by immunizing New Zealand White rabbits with the recombinant soluble 4 domain, CD4) or a mouse MAb against CCR5 (CTC5; R&D Systems, Minneapolis, Minn.).

Techniques: Expressing, Immunohistochemistry, Staining, Marker, Confocal Microscopy

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: 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: Frontiers in Immunology

Article Title: Melanocortin 1 Receptor Deficiency in Hematopoietic Cells Promotes the Expansion of Inflammatory Leukocytes in Atherosclerotic Mice

doi: 10.3389/fimmu.2021.774013

Figure Lengend Snippet: | MC1-R is expressed on splenic CD4 + T cells and its deficiency selectively modulates CCR5 expression. (A) Immunofluorescence staining of MC1-R and CD4 in the mouse spleen. White arrows indicate co-localization of MC1-R and CD4. RP indicates red pulp; WP, white pulp. (B) Western blot analysis of MC1-R protein expression in isolated CD4 + T cell samples from the spleen. The expression of vinculin is shown as loading control. (C) Quantitative real-time PCR (qPCR) analysis of chemokine receptor and adhesion molecule expression in isolated CD4 + T cells from Apoe -/- or Apoe -/- Mc1r e/e mice. Lanes on the right were incubated in anti-Mc1r antibody solution that was premixed with a molar excess of a blocking Mc1r peptide (D, E) Representative Western blots and quantification of CCR5 and β-actin (loading control) in isolated CD4 + T cells lysates from Apoe -/- or Apoe -/- Mc1r e/e mice. (F) Quantification of CCR5 surface expression by flow cytometry in CD4 + and CD8 + T cells from the spleen of Apoe -/- or Apoe -/- Mc1r e/e mice. Data are mean ± SEM, *P < 0.05, **P < 0.01 versus control Apoe -/- mice. Each dot represents individual mouse.

Article Snippet: Blots were probed with antibodies against CCR5 (Novus Biologicals, Bio-techne Ltd, UK) and MC1-R (Alomone Labs, Jerusalem, Israel).

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Isolation, Control, Real-time Polymerase Chain Reaction, Incubation, Blocking Assay, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Melanocortin 1 Receptor Deficiency in Hematopoietic Cells Promotes the Expansion of Inflammatory Leukocytes in Atherosclerotic Mice

doi: 10.3389/fimmu.2021.774013

Figure Lengend Snippet: MC1-R deficient CD4 + T cells show impaired CCR5-dependent migration and recycling of the CCR5 receptor. (A–C) Transwell migration assay using splenocytes from Apoe -/- and Apoe -/- Mc1r e/e mice. Cell were allowed to migrate for 3 hours towards the indicated chemokines and migrated CD4 + T cells, CD8 + T cells and Ly6C high monocytes were quantified by flow cytometry as percentage of input CD45 + cells. n=4 mice per genotype (D, E) Quantification of CCR5 surface expression on CD4 + cells and CD8 + T cells that were isolated from Apoe -/- or Apoe -/- Mc1r e/e mice. CCR5 surface expression was quantified during baseline, after CCL5-induced internalization and after withdrawal of CCL5. n=4 mice per genotype. Data are mean ± SEM., *P < 0.05, **P < 0.01 versus Apoe -/- mice.

Article Snippet: Blots were probed with antibodies against CCR5 (Novus Biologicals, Bio-techne Ltd, UK) and MC1-R (Alomone Labs, Jerusalem, Israel).

Techniques: Migration, Transwell Migration Assay, Flow Cytometry, Expressing, Isolation