anti cxcl12 neutralizing antibody  (R&D Systems)

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) GSEA plots of “chemokine signaling pathway” and “chemotaxis” gene sets enriched in MAOB-OE PrSC cells compared with controls. ( B ) ELISA of CXCL12 secretion in the culture media of control and MAOB-KD PrSC cells ( n = 3). ( C ) Representative IHC images and corresponding Pearson’s correlation analysis of stroma-expressed MAOB and CXCL12 protein levels in a PC TMA ( n = 37). Scale bars, 100 μm. ( D ) Pearson’s correlation analysis of MAOB and CXCL12 mRNA levels in patient-derived cultured prostatic stromal cells (left, n = 20) and laser-capture microdissected breast tumor stroma (right, n = 53) from GSE34312 and GSE9014 datasets, respectively. Statistical analysis was performed using one-way ANOVA with Dunnett’s test in (B). Data represent means ± SEM. ** P < 0.01.

Article Snippet: For determining the effect of stromal-derived MAOB/CXCL12-CXCR4 paracrine signaling on tumor cell proliferation in cocultures, selegiline, anti-CXCL12-neutralizing antibody (R&D Systems, MAB310, RRID:AB_2276927), rCXCL12 protein, or AMD3100 was added to cocultures 24 hours after tumor cell seeding, followed by determination of tumor cell proliferation.

Techniques: Chemotaxis Assay, Enzyme-linked Immunosorbent Assay, Control, Derivative Assay, Cell Culture

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) Western blot of Twist1 in control and MAOB-manipulated PrSC cells upon NAC (5 mM, 48 hours) or H 2 O 2 (40 μM, 24 hours) treatment. ( B ) ELISA of CXCL12 secretion in culture media of control and MAOB-OE PrSC cells treated with TWIST1 siRNA or NAC (5 mM, 48 hours) ( n = 3). ( C ) qPCR of CXCL12 in indicated PrSC cells upon NAC treatment (5 mM, 48 hours) or Twist1/ TWIST1 siRNA expression ( n = 3). ( D and E ) Determination of CXCL12 mRNA (D) and 0.7-kb promoter activity (E) in PrSC cells upon Twist1 expression and/or TGFβ1 treatment (10 ng/ml, 12 hours) ( n = 3). ( F ) Schematic diagrams of WT and mutated CXCL12 E-box/SBE-Luc constructs and determination of their activities in PrSC cells upon Twist1 expression and/or TGFβ1 treatment (10 ng/ml, 12 hours) ( n = 3). ( G ) Representative proximity ligation assay staining and quantitation of indicated Twist1-Smad interactions by per-nucleus fluorescence intensity in control and MAOB-OE PrSC cells. Smad antibody incubation alone served as negative control. Numbers of nuclei included for comparisons between groups are denoted. Scale bars, 50 μm. ( H ) Co-IP assays of indicated Twist1-Smad interactions in PrSC cells with coexpression of Twist1 and individual Smads. Immunoglobulin G (IgG) was used in IP as negative control. Ten percent input was blotted as positive control. ( I ) ChIP analysis of chromatin from control and MAOB-OE PrSC cells precipitated with anti-Twist1, anti-Smad4, or a control IgG, followed by qPCR probing the E-box/SBE-centric CXCL12 promoter region ( n = 3). ( J ) ChIP analysis of chromatin from PrSC cells precipitated with anti-Smad4 antibody and then reprecipitated with anti-Twist1 or a control IgG (re-ChIP), followed by qPCR probing the E-box/SBE-encompassing CXCL12 promoter sequence ( n = 3). Statistical analysis was performed using one-way ANOVA with Tukey’s test. Data represent means ± SEM. * P < 0.05, ** P < 0.01; ns, not significant.

Article Snippet: For determining the effect of stromal-derived MAOB/CXCL12-CXCR4 paracrine signaling on tumor cell proliferation in cocultures, selegiline, anti-CXCL12-neutralizing antibody (R&D Systems, MAB310, RRID:AB_2276927), rCXCL12 protein, or AMD3100 was added to cocultures 24 hours after tumor cell seeding, followed by determination of tumor cell proliferation.

Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay, Expressing, Activity Assay, Construct, Proximity Ligation Assay, Staining, Quantitation Assay, Fluorescence, Incubation, Negative Control, Co-Immunoprecipitation Assay, Positive Control, Sequencing

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) Quantitation of C4-2 and PC-3 PC cells in monoculture and coculture with indicated PrSC fibroblasts upon anti-CXCL12 (0.1 μg/ml) antibody treatment ( n = 3). ( B ) Quantitation of PC cells in coculture with indicated PrSC cells treated with rCXCL12 protein (50 ng/ml; n = 3). ( C ) Western blot of CXCR4 and CXCR7 in control and CXCR4-KD/CXCR7-KD PC cells. ( D and E ) Quantitation of control and CXCR4-KD/CXCR7-KD PC cells in coculture with indicated PrSC cells treated without (D) or with (E) rCXCL12 (50 ng/ml; n = 3). ( F ) Quantitation of PC cells in coculture with indicated PrSC cells treated with 10 nM AMD3100 ( n = 3). ( G ) Representative images and quantitation of PC-3 cell migration and invasion in coculture with indicated PrSC cells upon treatment with anti-CXCL12 antibody (0.1 μg/ml) or 10 nM AMD3100 ( n = 3). Scale bars, 200 μm. ( H ) Representative images and quantitation of PC cell migration in coculture with indicated PrSC cells treated with rCXCL12 (50 ng/ml; n = 3). Scale bars, 100 μm. ( I ) Phospho-antibody array analysis of PC-3 cells treated with indicated PrSC cell CM. All phosphoprotein levels were normalized to their total forms from a single array with six replicate spots, with significantly activated phosphoproteins (fold change > 1.5, P < 0.05) denoted. ( J ) Western blot of p-Src and p-JNK in control and CXCR4-KD PC-3 cells treated with indicated PrSC cell CM. ( K ) Western blot of p-Src and p-JNK in PC-3 cells treated with indicated PrSC cell CM plus rCXCL12 (50 ng/ml). ( L ) Quantitation of PC cells in coculture with indicated PrSC cells following pretreatment with 40 nM Src inhibitor 1 or 10 μM SP600125 for 24 hours ( n = 3). Statistical analysis was performed using one-way ANOVA with Tukey’s test. Data represent means ± SEM. * P < 0.05, ** P < 0.01; ns, not significant.

Article Snippet: For determining the effect of stromal-derived MAOB/CXCL12-CXCR4 paracrine signaling on tumor cell proliferation in cocultures, selegiline, anti-CXCL12-neutralizing antibody (R&D Systems, MAB310, RRID:AB_2276927), rCXCL12 protein, or AMD3100 was added to cocultures 24 hours after tumor cell seeding, followed by determination of tumor cell proliferation.

Techniques: Quantitation Assay, Western Blot, Control, Migration, Ab Array

Journal: Science Advances

Article Title: Stromal-derived MAOB promotes prostate cancer growth and progression

doi: 10.1126/sciadv.adi4935

Figure Lengend Snippet: ( A ) Quantitation of C4-2 and PC-3 cell proliferation in monoculture and coculture with control and MAOB-OE PrSC cells upon selegiline treatment (10 nM, 72 hours) ( n = 3). ( B ) BLI-based growth curves of Luc-tagged PC-3 tumors grown in the prostates of male NSG mice treated with selegiline at various doses (0.5, 2, and 10 mg/kg) or saline as a vehicle ( n = 5 per group). ( C ) BLI images of mice from each group at the end point. ( D ) Determination of tumor weights ( n = 5). ( E ) Determination of MAOA and MAOB enzymatic activities in mouse liver tissue from each group at the end point ( n = 3). ( F ) Representative images of H&E and IHC staining of tumor-expressed Ki-67, p-Src, and p-JNK and stroma-expressed αSMA and CXCL12 and their quantitation in tumor samples from each group ( n = 5). Scale bars, 100 μm. ( G ) Mouse body weights determined weekly ( n = 5). ( H ) Representative H&E images of mouse liver and kidney tissue from each group. Scale bars, 100 μm. ( I to L ) ELISA of ALT (I), AST (J), BUN (K), and creatinine (L) in mouse sera at the end point ( n = 5). ( M ) Schematic depicting stromal-derived MAOB activation of paracrine CXCL12-CXCR4/Src/JNK signaling through interplay between ROS-dependent Twist1 (via a HIF1α/VEGF-A/AKT/FOXO1 pathway) and TGFβ1/Smads to promote stromal-epithelial interactions for PC growth and progression. Statistical analysis was performed using one-way ANOVA with Tukey’s test in (A), (D) to (F), and (I) to (L) and two-way ANOVA with Tukey’s test in (B) and (G). Data represent means ± SEM. * P < 0.05, ** P < 0.01; ns, not significant.

Article Snippet: For determining the effect of stromal-derived MAOB/CXCL12-CXCR4 paracrine signaling on tumor cell proliferation in cocultures, selegiline, anti-CXCL12-neutralizing antibody (R&D Systems, MAB310, RRID:AB_2276927), rCXCL12 protein, or AMD3100 was added to cocultures 24 hours after tumor cell seeding, followed by determination of tumor cell proliferation.

Techniques: Quantitation Assay, Control, Saline, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Derivative Assay, Activation Assay

Journal: Frontiers in Oncology

Article Title: CD62L dim Neutrophils Specifically Migrate to the Lung and Participate in the Formation of the Pre-Metastatic Niche of Breast Cancer

doi: 10.3389/fonc.2020.540484

Figure Lengend Snippet: CD62L dim neutrophils initially aggregate in the lung by expressing CXCR4 at high levels, (A) RT-PCR analysis of chemokine receptor expression in CD62L dim and CD62L hi neutrophils. β-Actin expression in CD62L hi neutrophils was assayed as a control. (B) MFI of CXCR4 expression on CD62L dim and CD62L hi neutrophils from tumor-bearing mice, as analyzed using flow cytometry. (C) Flow cytometry analysis of the chemotaxis of cells induced by rCXCL12 after a 1:1 mixture of CD62L dim and CD62L hi neutrophils were seeded in the upper chamber. (D) Fluorescence imaging of labeled neutrophils after rCXCL12 chemotaxis. (E) RT-PCR analysis of Cxcl12 expression in various organs of naïve mice and 2-week tumor-bearing mice. (F) Flow cytometry analysis of the proportion of neutrophils after chemotaxis using the lung tissue culture supernatant (LTCS) of cells from tumor-bearing mice. (G) Flow cytometry analysis of the number of CD62L dim neutrophils after chemotaxis using the LTCS of cells from tumor-bearing mice in the presence or absence of the anti-CXCL12 mAb. Data are presented as the mean ± SEM of one representative experiment. Similar results were obtained in three independent experiments. Unpaired Student’s t tests, ns, not significant. *p < 0.05 and ***p < 0.001. See also .

Article Snippet: Medium supplemented with recombinant murine CXCL12 (100 ng/mL, Peprotech, #250-20A), lung tissue culture supernatant (LTCS), or a CXCL12 neutralizing antibody (200 μg/mL, R&D Systems, #MAB310-SP, clone: 79014) was added to the remaining receiver wells.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Flow Cytometry, Chemotaxis Assay, Fluorescence, Imaging, Labeling