Journal: PLoS ONE

Article Title: Differential Estrogen-Regulation of CXCL12 Chemokine Receptors, CXCR4 and CXCR7, Contributes to the Growth Effect of Estrogens in Breast Cancer Cells

doi: 10.1371/journal.pone.0020898

Figure Lengend Snippet: Cells were cultured as described in “ ” and treated with E2 or ICI 184,780 (ICI) using EtOH as vehicle. The vehicle-only treatment served as a control. The mRNA levels of CXCL12 (A and B), CXCR4 (D and E) and CXCR7 (G and H) were quantified by real-time PCR analysis of cells treated for different periods of time with 10 −8 M E2 (A, D and G) or cells treated for 48 h with 10 −8 M E2 alone, 10 −6 M ICI alone or both E2 and ICI (B, E and H). The real-time PCR results were normalized against the internal control GAPDH and expressed as the mean CXCL12 , CXCR4 or CXCR7/GAPDH mRNA ratio ± SEM of at least three independent experiments. Protein levels of CXCL12 (C), CXCR4 (F) and CXCR7 (I) was assayed. Secreted CXCL12 protein levels after treatment with EtOH or 10 −8 M E2 for 48 h were determined by ELISA, and the values were normalized relative to the total protein concentration (C). The expression of CXCR4 and CXCR7 at the surface of MCF-7 cells was measured by flow cytometry after treatment with EtOH or 10 −8 M E2 for 48 h (F, I). Representative data from at least three experiments performed in duplicate are shown. Asterisks or different lowercase letters indicate significant differences ( p <0.05) between the control and treated cells.

Article Snippet: The antibodies used for FACS were murine monoclonal (Mm) antibody anti-human CXCR4 (clone 12G5, R&D Systems) and Mm antibody anti-human CXCR7/RDC1 (clone 11G8, R&D Systems, Minneapolis, MN, USA).

Techniques: Cell Culture, Control, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Protein Concentration, Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: Differential Estrogen-Regulation of CXCL12 Chemokine Receptors, CXCR4 and CXCR7, Contributes to the Growth Effect of Estrogens in Breast Cancer Cells

doi: 10.1371/journal.pone.0020898

Figure Lengend Snippet: CXCL12 , CXCR4 and CXCR7 mRNA were assessed by quantitative real time PCR after 48 h treatment of ZR-75 (A) or MDA-MB-231 (B) cells to EtOH (−) or to10 −8 M E2 (+). Transcript levels were normalized against GAPDH mRNA and data were calculated as percentage of the E2 effect. Data are from triplicate samples and are representative of three separate experiments. Asterisk indicates significant differences ( p <0.05) between the control and ligand treated cells.

Article Snippet: The antibodies used for FACS were murine monoclonal (Mm) antibody anti-human CXCR4 (clone 12G5, R&D Systems) and Mm antibody anti-human CXCR7/RDC1 (clone 11G8, R&D Systems, Minneapolis, MN, USA).

Techniques: Real-time Polymerase Chain Reaction, Control

Journal: PLoS ONE

Article Title: Differential Estrogen-Regulation of CXCL12 Chemokine Receptors, CXCR4 and CXCR7, Contributes to the Growth Effect of Estrogens in Breast Cancer Cells

doi: 10.1371/journal.pone.0020898

Figure Lengend Snippet: The levels of the CXCL12, CXCR4 and CXCR7 transcripts were assessed by quantitative real-time PCR in MCF-7 cells treated under various conditions for 48 h. Treatment with EtOH and 10 −8 M E2 served as the negative and positive controls, respectively. In each experimental assay, the cells were exposed to different concentrations of 17 α-ethynyl-estradiol (EE2) (A) and Genistein (Gen) (B). Transcript levels were normalized against GAPDH mRNA, and data were calculated as percentage of the E2 effect for each experiment. Significant differences (P<0.05) are indicated by different lowercase letters.

Article Snippet: The antibodies used for FACS were murine monoclonal (Mm) antibody anti-human CXCR4 (clone 12G5, R&D Systems) and Mm antibody anti-human CXCR7/RDC1 (clone 11G8, R&D Systems, Minneapolis, MN, USA).

Techniques: Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Differential Estrogen-Regulation of CXCL12 Chemokine Receptors, CXCR4 and CXCR7, Contributes to the Growth Effect of Estrogens in Breast Cancer Cells

doi: 10.1371/journal.pone.0020898

Figure Lengend Snippet: (A) FAIRE assays were performed on MCF-7 cells exposed to either ETOH (−) or 10 −8 M E2 (+) for 48 h. Real-time PCR was performed to monitor enrichment of the DNA corresponding to the proximal promoters of the CXCL12 , CXCR4 and CXCR7 genes relative to input chromatin. The data are from triplicate samples and are representative of three separate experiments. Asterisks indicate significant differences ( p <0.05) between the control and treated cells. (B) The Integrated Genome Browser (Affymetrix) was used to visualize ER-binding sites in the regions surrounding the CXCL12 , CXCR4 and CXCR7 genes. Raw ChIP-chip data for ER and high confidence ER-binding sites called using the MAT algorithm are shown , . The numbered ER-binding sites correspond to bound regions in which the closest TSS is that of CXCL12 , CXCR4 or CXCR7 . Arrows indicate the orientation of the CXCL12 , CXCR4 and CXCR7 genes.

Article Snippet: The antibodies used for FACS were murine monoclonal (Mm) antibody anti-human CXCR4 (clone 12G5, R&D Systems) and Mm antibody anti-human CXCR7/RDC1 (clone 11G8, R&D Systems, Minneapolis, MN, USA).

Techniques: Real-time Polymerase Chain Reaction, Control, Binding Assay, ChIP-chip

Journal: PLoS ONE

Article Title: Differential Estrogen-Regulation of CXCL12 Chemokine Receptors, CXCR4 and CXCR7, Contributes to the Growth Effect of Estrogens in Breast Cancer Cells

doi: 10.1371/journal.pone.0020898

Figure Lengend Snippet: siRNA directed against CXCL12, CXCR4 or CXCR7 was transfected into MCF-7 cells treated with EtOH (−) or 10 −8 M E2 (+). (A) After 48 h, the levels of the CXCL12, CXCR4 and CXCR7 transcripts were assessed by quantitative PCR and normalized against GAPDH mRNA. The results were compared with those obtained from MCF-7 cells transfected with a nonspecific siRNA control. (B) Total protein was extracted from MCF-7 cells, and the levels of CXCL12, CXCR4 and CXCR7 were analyzed by Western blotting. (C) To determine the growth rate of the MCF-7 cells, the siRNA-transfected cells were treated with EtOH (−E2) or 10 −8 M E2 (+E2) for seven days. E2-dependent and -independent cell growth were evaluated using MTT assays of three independent experiments (n = 6). The results are expressed as a percentage of the relative cell number obtained from cells transfected with the control siRNA and treated with EtOH (considered as 100%). Significant differences ( p <0.05) between transfected cells in the absence of E2 are indicated by an asterisk and between transfected cells in the presence of E2 by a sharp symbol. (D) The effects of specific inhibitors for CXCL12 (Chalcon 4), CXCR4 (AMD3100) or CXCR7 (CCX771) were measured after treatment of MCF-7 cells with either EtOH (−E2) or 10 −8 M E2 (+E2) for 7 days. DMSO (vehicle) was used as the control. E2-dependent and E2-independent cell growth were then evaluated by MTT assays of three independent experiments (n = 6). The results are expressed as a percentage of the relative cell number obtained from cells treated with the vehicle control (considered as 100%). Significant differences ( p <0.05) between treated cells in the absence of E2 are indicated by an asterisk and between treated cells in the presence of E2 by a sharp symbol.

Article Snippet: The antibodies used for FACS were murine monoclonal (Mm) antibody anti-human CXCR4 (clone 12G5, R&D Systems) and Mm antibody anti-human CXCR7/RDC1 (clone 11G8, R&D Systems, Minneapolis, MN, USA).

Techniques: Transfection, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: PLoS ONE

Article Title: Differential Estrogen-Regulation of CXCL12 Chemokine Receptors, CXCR4 and CXCR7, Contributes to the Growth Effect of Estrogens in Breast Cancer Cells

doi: 10.1371/journal.pone.0020898

Figure Lengend Snippet: MCF-7 cells were transiently transfected with either a control expression vector or one containing the human CXCR7 open reading frame. (A) Total protein extracts were prepared 48 h after transfection, and a Western blot analysis was performed to confirm CXCR7 over-expression. (B) Transfected cells were cultured in the presence of EtOH (−) or 10 −8 M E2 (+) for seven days. E2-dependent and E2-independent cell growth rates were then evaluated by cell count of three independent experiments (n = 3). The results are expressed as a percentage of the relative cell number obtained from control cells treated with E2 (considered as 100%). Significant differences ( p <0.05) between transfected cells in the absence of E2 are indicated by an asterisk and between transfected cells in the presence of E2 by a sharp symbol. (C) A proposed model for the involvement of the CXCL12 signaling axis in E2-dependent and -independent cell growth is shown. The binding of CXCL12 to CXCR4 and CXCR7 leads to the stimulation of cell growth through diverse pathways . CXCR7 can also modulate CXCL12 availability by removing the chemokine from the extracellular space (left panel). Estrogens could stimulate cell growth by favoring the activation of CXCL12 through CXCR4 and reducing the expression of CXCR7 (right panel).

Article Snippet: The antibodies used for FACS were murine monoclonal (Mm) antibody anti-human CXCR4 (clone 12G5, R&D Systems) and Mm antibody anti-human CXCR7/RDC1 (clone 11G8, R&D Systems, Minneapolis, MN, USA).

Techniques: Transfection, Control, Expressing, Plasmid Preparation, Western Blot, Over Expression, Cell Culture, Cell Counting, Binding Assay, Activation Assay

Journal: Cell reports. Medicine

Article Title: Receptor transfer between immune cells by autoantibody-enhanced, CD32-driven trogocytosis is hijacked by HIV-1 to infect resting CD4 T cells.

doi: 10.1016/j.xcrm.2024.101483

Figure Lengend Snippet: Figure 2. Characterization of CD32-driven trogocytosis (A) 293T cells transiently expressing C-terminal GFP fusion proteins of FcgRs CD32A, CD32B, or CD32C or, as a control, the nucleocytoplasmic dNTPase SAMHD1 served as donors in co-cultures with CellTrace dye-stained SupT1 T target cells. All culture media contained IgG-depleted FCS. Shown are repre- sentative flow cytometry dot plots and the percentages of CD32+ and GFP+ target T cells. One experiment out of two is shown. (B) Schematic of topology determination of transferred CD32-GFP (top). Bottom: SupT1 T cells were co-cultured as described in (A) and stained with either an anti-GFP mAb or an isotype control antibody, both conjugated to Alexa 647, with or without prior cell permeabilization. One representative experiment is shown (n = 3). The illustration was created with BioRender.com. (C) 293T cells were co-transfected with plasmids encoding C-terminal GFP fusion proteins of CD32A, CD32B, or CD32C or, as a control, histone H2B-GFP, together with a plasmid encoding CCR5. After 2 days, cells were either left untreated or pre-treated with an anti-CD32 Ab or an isotype control Ab prior to co- cultivation with SupT1 T cells. One day later, the expression of GFP and CCR5 on the target T cells was determined by flow cytometry. Mean ± SEM are shown (n = 3). Asterisks indicate statistical significance by two-way ANOVA. p values were corrected for multiple comparison (Tukey). (D) Half-life of CD32 and CCR5 surface expression on SupT1 target cells following co-culture as in (A). Following 1 day of co-culture, SupT1 T cells positive for CD32-GFP were sorted by flow cytometry and kept in culture for an additional 9 days. The expression of CD32 (top) or CCR5 (bottom) on sorted cells was determined for up to 192 h of cultivation. One representative experiment is shown (n = 2). (E) Schematic of CD32B with important amino acids and motifs indicated. (F) Transfer of the indicated CD32B mutants, CD32A WT, CD32C WT, or H2B (GFP fusion proteins), assessed as in (A) (mean ± SEM; n = 4). Asterisks indicate statistical significance by one-way ANOVA. p values were corrected for multiple comparison (Dunnett). (G) Visualization of the material transfer from CD32B-GFP expressing 293T cells to LifeAct-mCherry-expressing SupT1 using live-cell imaging. 293T cells transiently expressing CD32B-GFP (green) were co-cultured with LifeAct-mCherry-expressing SupT1 cells (magenta), cultivated in IgG-depleted FCS and boosted with PGT151 antibody, and imaged using spinning disc microscopy for 4 h. The left panel shows the beginning of co-culture. (a) Labels the area with the first transfer event (middle panel). (b) Labels the area of the second transfer event (right panel). Dashed white box marks the area that is zoomed and depicted with individual time points before and after the transfer event (shown below). The time stamp (upper right corner, relative to the time frame which shows the transfer event (time 00:00) in zoom-ins). Scale bar, 10 mm. *p % 0.05; **p % 0.01; ***p % 0.001.

Article Snippet: Human T cell line SupT1 (DSMZ, ACC 140) was cultivated in RPMI 1640 GlutaMAX (Gibco) supplemented with 10% (v/v) FBS and Penicillin-Streptomycin (100 IU/mL).

Techniques: Expressing, Control, Staining, Cytometry, Cell Culture, Transfection, Plasmid Preparation, Comparison, Co-Culture Assay, Live Cell Imaging, Microscopy

Journal: Cell reports. Medicine

Article Title: Receptor transfer between immune cells by autoantibody-enhanced, CD32-driven trogocytosis is hijacked by HIV-1 to infect resting CD4 T cells.

doi: 10.1016/j.xcrm.2024.101483

Figure Lengend Snippet: Figure 3. CD32-driven trogocytosis is boosted by T cell-autoreactive antibodies associated with chronic HIV-1 infection (A) CD32 expression on CD4 T cells from peripheral blood of healthy donors (HD) (n = 23) and chronic HIV-1 infected patients (CHI) (n = 39). Median with 95% CI are shown. Asterisks indicate statistical significance by Mann-Whitney test. (B) 293T cells transiently co-expressing CD32B-GFP and CCR5 were pre-treated with the indicated patient sera before 1 day of co-culture with SupT1 T cells. Shown are the percentage of CD32B-GFP+ and CCR5+ target cells (median with 95% CI, each dot represents a different patient; see also Figure S7C). CHI, chronic HIV-1 infection; ART, anti-retroviral therapy; AHI, acute HIV-1 infection. Fiebig stages II-III of acute HIV-1 infection42; HIV-2, HIV type 2; HTLV-1, human T cell lymphotropic virus type 1; HCV, hepatitis C virus; DENV, dengue virus; YFV, yellow fever virus-vaccinated; SARS-CoV-2, severe acute respiratory syndrome coronavirus type 2; EC, Echinococcus multilocularis; SCH, Schistosoma spp.; TB, Mycobacterium tuberculosis; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; CG, cryoglobulinemia. Asterisks indicate statistical significance by Mann-Whitney test. (C) Percentage of GFP+ target cells after 1 day of co-culture with 293T cells as in (B). IgG was depleted from the sera of two healthy donor (HD) and two HIV-1 patient (CHI) samples from (B, pink and red) and input (original sera), flowthrough and eluate of the IgG depletion were used for pre-treatment of cells prior to co- culture. Mean of two donors from each category is shown. (D) Correlation of antibody binding to SupT1 T cells and CD32B-GFP trogocytosis as in (B), with sera from HIV-1 patients. P, Pearson correlation coefficient. (E) Binding of sera with high or low trogocytotic activity (pink and red dots in B) to primary CD4 T cells as detected with fluorochrome-coupled anti-human IgG Ab (median with 95% CI, CD4 T cells; n = 3). Kruskal-Wallis test with Dunn’s multiple-testing correction. (F) A panel of bNAbs was analyzed for binding to uninfected resting CD4 T cells (top) or activated CD4 T cells (bottom). Mean ± SEM; n = 3. Asterisks indicate statistical significance by one-way ANOVA (top) or three-way ANOVA (bottom). p values were corrected for multiple comparison (Dunnett). (G) Purified, CMV-encoded, soluble Fc-binding proteins gp34 and gp68, or control proteins gp34 non-binding mutant (mtrp; W65F) and soluble ICOSL (inducible T cell co-stimulator ligand) were added to 293T donor cells as in (A), in the presence of PGT151 Ab, and subsequently co-cultured with SupT1 T cells. CD32 transfer was evaluated as in (B). Asterisks indicate statistical significance by two-way ANOVA. p values were corrected for multiple comparison (Tukey). *p % 0.05, **p % 0.01, ***p % 0.001; n.s., not significant. (H) Schematic of the determinants of antibodies for trogocytosis enhancement.

Article Snippet: Human T cell line SupT1 (DSMZ, ACC 140) was cultivated in RPMI 1640 GlutaMAX (Gibco) supplemented with 10% (v/v) FBS and Penicillin-Streptomycin (100 IU/mL).

Techniques: Infection, Expressing, MANN-WHITNEY, Co-Culture Assay, Retroviral, Virus, Binding Assay, Activity Assay, Comparison, Control, Mutagenesis, Cell Culture

Journal: Viruses

Article Title: A Novel CXCR4 Targeting Protein SDF-1/54 as an HIV-1 Entry Inhibitor

doi: 10.3390/v11090874

Figure Lengend Snippet: Capability of SDF-1/54 inducing CXCR4 internalization. MT-2 cells were treated with AMD3100 and SDF-1/54 for 2 h at 50 nM and CXCR4 on the cell surface was detected indirectly by PE-labeled CXCR4 antibody using flow cytometry. The level of CXCR4 on the cell surface is shown as mean fluorescence density, as described in Materials and Methods. MT-2 cells without any treatment were used as a control.

Article Snippet: PE-conjugated mouse anti-human CXCR4 (clone12G5) antibody was purchased from R&D Systems (USA).

Techniques: Labeling, Flow Cytometry, Fluorescence, Control

Journal: PLoS ONE

Article Title: Reassessment of CXCR4 Chemokine Receptor Expression in Human Normal and Neoplastic Tissues Using the Novel Rabbit Monoclonal Antibody UMB-2

doi: 10.1371/journal.pone.0004069

Figure Lengend Snippet: A , Western blot analysis of the specificity of anti-CXCR4 antibodies. Membrane preparations from HEK-293 cells stably transfected to express either CCR7 or CXCR4 were separated on 10% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes. Membranes were then incubated with affinity-purified 1 µg/ml anti-CCR7 {1188} or anti-CXCR4 {UMB-2} hybridoma supernatant at a dilution of 1∶100. Blots were developed using enhanced chemiluminescence. Note that UMB-2 detected a band of the expected molecular weight only in CXCR4- but not in CCR7-transfected cells. Two additional experiments gave similar results. Ordinate , migration of protein molecular weight markers ( M r ×10 −3 ). B , characterization of UMB-2 by immunofluorescent staining of transfected cells. HEK-293 cells expressing CCR7 or CXCR4 were either not exposed or exposed to 100 ng/ml MIP-3 or 100 ng/ml SDF-1 for 30 min, subsequently fixed and immunofluorescently stained with 1 µg/ml anti-CCR7 {1188} or anti-CXCR4 {UMB-2} at a dilution of 1∶100. Note that UMB-2 detected prominent immunofluorescence at the level of the plasma membrane only in CXCR4- but not in CCR7-expressing cells, and that SDF-1 exposure induced a rapid translocation of CXCR4 receptor immunostaining from the plasma membrane into the cytosol. Representative results from one of three independent experiments are shown. Scale bar, 20 µm.

Article Snippet: The mouse monoclonal anti-CXCR4 antibodies {12G5} and {44716} were obtained from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, Membrane, Stable Transfection, Transfection, Incubation, Affinity Purification, Molecular Weight, Migration, Staining, Expressing, Immunofluorescence, Clinical Proteomics, Translocation Assay, Immunostaining

Journal: PLoS ONE

Article Title: Reassessment of CXCR4 Chemokine Receptor Expression in Human Normal and Neoplastic Tissues Using the Novel Rabbit Monoclonal Antibody UMB-2

doi: 10.1371/journal.pone.0004069

Figure Lengend Snippet: A , Western blot analysis of the specificity of UMB-2 using brain homogenates from CXCR4-deficient mice (CXCR4−/−) prepared at embryonic day 17 (E17) and their wild-type littermates (CXCR4+/+) as well adult CXCR4+/+ brain prepared at postnatal day 90 (P90). Note that UMB-2 selectively detects CXCR4 receptors at E17 in CXCR4+/+ mice and does not cross-react with other proteins present in tissue extracts prepared from CXCR4−/− mice. Migration of protein molecular weight markers ( M r ×10 −3 ) is indicated on the right. B–E , characterization of UMB-2 by immunohistochemical staining of formalin-fixed, paraffin-embedded brain sections prepared at E17 from CXCR4-deficient mice (CXCR4−/−, left panel ) and their wild-type littermates (CXCR4+/+, right panel ). Note that UMB-2 selectively detects CXCR4 receptors and does not cross-react with other proteins present in tissue sections from CXCR4−/− mice. F , prominent CXCR4 receptor immunostaining at the plasma membrane of tangentially migrating neurons in the cortex. CTX, cortex; GE, ganglionic eminence; CPu, caudate putamen; OX, optic chiasm; 3V, third ventricle; MZ, marginal zone; CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Scale bars, B = C = 500 µm, D = E = 100 µm, F = 50 µm.

Article Snippet: The mouse monoclonal anti-CXCR4 antibodies {12G5} and {44716} were obtained from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, Migration, Molecular Weight, Immunohistochemical staining, Staining, Formalin-fixed Paraffin-Embedded, Immunostaining, Clinical Proteomics, Membrane

Journal: PLoS ONE

Article Title: Reassessment of CXCR4 Chemokine Receptor Expression in Human Normal and Neoplastic Tissues Using the Novel Rabbit Monoclonal Antibody UMB-2

doi: 10.1371/journal.pone.0004069

Figure Lengend Snippet: A – K , UMB-2 immunohistochemical staining of a variety of human normal and neoplastic tissues. Sections were dewaxed, microwaved in citric acid and incubated with UMB-2 at a dilution of 1∶10. Sections were sequentially treated with biotinylated anti-rabbit IgG and AB solution. Sections were then developed in diaminobenzidine and lightly counterstained with hematoxylin. Note that UMB-2 detected CXCR4 receptors at the plasma membrane of a subset of tumor cells in a variety of human tumors including mammary carcinoma (A), endometrial carcinoma (B), cervical carcinoma (C), ovarian carcinoma (D), gastric carcinoma (E), pancreatic carcinoma (F), colonic carcinoma (G) and malignant pheochromocytoma (J). In nearly all of these cases UMB-2 immunoreactive tumor cells exhibited a heterogeneous distribution throughout the tumor. UMB-2 revealed predominant cytoplasmic staining in prostate carcinoma (H). In glioblastoma, UMB-2 detected CXCR4 receptors predominantly on the plasma membrane of endothelial cell of tumoral blood vessels (I). UMB-2 also detected CXCR4 receptors in the zona fasciculata of the adrenal cortex (K). Inset in C, peptide adsorption control. Arrowheads in I, tumoral blood vessels. C, capsule; Z.g., Zona glomerulosa; Z.f., Zona fasciculata; Z.r., Zona reticularis. Scale bars, A = B = C = D = E = F = G = H = I = J = K = 50 µm, K = 100 µm.

Article Snippet: The mouse monoclonal anti-CXCR4 antibodies {12G5} and {44716} were obtained from R&D Systems (Minneapolis, MN).

Techniques: Immunohistochemical staining, Staining, Incubation, Clinical Proteomics, Membrane, Adsorption, Control

Journal: PLoS ONE

Article Title: Reassessment of CXCR4 Chemokine Receptor Expression in Human Normal and Neoplastic Tissues Using the Novel Rabbit Monoclonal Antibody UMB-2

doi: 10.1371/journal.pone.0004069

Figure Lengend Snippet: Prevalence of CXCR4 receptors in human tumors.

Article Snippet: The mouse monoclonal anti-CXCR4 antibodies {12G5} and {44716} were obtained from R&D Systems (Minneapolis, MN).

Techniques: