recombinant human ephb4 fc chimera protein  (R&D Systems)

Journal: Cancer Research

Article Title: Inhibition of Ephrin B2 Reverse Signaling Abolishes Multiple Myeloma Pathogenesis

doi: 10.1158/0008-5472.CAN-23-1950

Figure Lengend Snippet: Inhibition of ephrin B2 suppresses multiple myeloma growth in vivo . A, Representative immunofluorescence images of RPMI-8226 multiple myeloma (MM) cells stained for BCMA (green) and EFNB2; merged image with DAPI (blue) nuclear stain at right (red). Scale bar, 100 μm. B, Representative immunofluorescence images of VE-cadherin (Cdh5) expression (white) and Ephb4 colocalization (green) in BM blood vessels in NSG mice. Scale bar, 100 μm. C, Representative images of femur cross sections from NSG mice at 5 weeks posttransplant of RPMI-8226 multiple myeloma cells; sinusoidal blood vessel expressing VE-cadherin (Cdh5; white), multiple myeloma cells expressing BCMA (green), and EFNB2 (red) and merged image shown at right. Scale bar, 100 μm. Arrows, multiple myeloma cells adjacent to sinusoidal vessel. D, Left, representative flow cytometric analysis of human CD138 + multiple myeloma cells in the BM of NSG mice at 5 weeks posttransplant of 5 × 10 6 RPMI-8226 cells treated with sh-EFNB2 or sh-Control . SSC, side scatter. Right, mean percentages of CD138 + multiple myeloma cells in the BM of transplanted mice. n = 8/group. E, Percent survival of NSG mice after transplantation of 5 × 10 6 RPMI-8226 cells transduced with sh-EFNB2 or sh-Control . Log-rank test, P = 0.0012, n = 12/group. F, Mean percentages of Annexin V + RPMI-8226 cells at 24 hours posttreatment with EFNB2 scFv ( n = 6/group). G, Top, schematic representation of experiment. Bottom left, representative flow cytometric analysis of human CD138 + multiple myeloma cells in the BM of NSG mice at 4 weeks posttransplantation of 3 × 10 6 RPMI-8226 cells and intravenous administration of EFNB2 scFv or IgG on days +4, 6, 8, 10, and 12. Bottom right, mean percentages of CD138 + multiple myeloma cells in the BM of transplanted mice. One-way ANOVA was utilized for analyses of three or more groups; Student two-sided t test was utilized otherwise. Error bars, SD. **, P < 0.01; ****, P < 0.0001; # , P < 0.05; ### , P < 0.001. ( G, Created with BioRender.com.)

Article Snippet: Using a 1:1 ratio, human recombinant EPHB4 Fc (R&D Systems, 11307-B4) was preclustered with human IgG1 mAb for 30 minutes at room temperature.

Techniques: Inhibition, In Vivo, Immunofluorescence, Staining, Expressing, Control, Transplantation Assay, Transduction

Journal: Cancer Research

Article Title: Inhibition of Ephrin B2 Reverse Signaling Abolishes Multiple Myeloma Pathogenesis

doi: 10.1158/0008-5472.CAN-23-1950

Figure Lengend Snippet: The effects of ephrin B2 on multiple myeloma are mediated by STAT5. A, Heat map of differentially expressed genes by RNA-seq of sh-EFNB2 versus sh-Control cells. B, Upstream regulator analysis from IPA of RNA-seq data. C, Selected STAT5 target genes in the RNA-seq data. D, Left, representative analysis of phospho-STAT5 in RPMI-8226 cells treated with sh-EFNB2 or sh-Control in presence of EPHB4-Fc. Right, mean fluorescence intensity (MFI) of pSTAT5 in the sh-EFNB2 - and sh-Control –treated multiple myeloma cells. E, Numbers of U266 cells in culture over time when transduced with gRNA1-EFNB2 and gRNA2-EFNB2 . F, Left, representative histograms of BrdU incorporation over 6 hours in U266 cells transduced with the labeled construct. Right, %BrdU + cells in each group. G, Left, representative flow cytometric analysis of Annexin V + 7AAD + (necrotic) and Annexin V + 7AAD − (apoptotic) cells. Right, mean levels of total Annexin V + cells within each treatment group. H, Mean percentages of Annexin V + U266 cells engineered with the indicated construct and exposed to bortezomib (Bor) or dexamethasone (Dex) for 24 hours. n = 6/group. I, Representative histograms of p-STAT5 in U266 cells transduced with gRNA-EFNB2 or gRNA-control , with and without stimulation by EPHB4-Fc. IgG (isotype) was used as a negative control for EPHB4-Fc. Right, mean MFI of pSTAT5 in each treatment population. J, Multiple myeloma cells transduced with gRNA-EFNB2 or gRNA-control were cultured with increasing concentrations of the STAT5 inhibitor 2-[(4-oxo-4H-1-benzopyran-3-yl)methylene]hydrazide 3-pyridinecarboxylic acid (STAT5i) for 24 hours, then BrdU incorporation analysis was performed. n = 6/group. One-way ANOVA used for analyses in which there were three or more groups, Student two-sided t test for the other analyses. Error bars, SD. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: Using a 1:1 ratio, human recombinant EPHB4 Fc (R&D Systems, 11307-B4) was preclustered with human IgG1 mAb for 30 minutes at room temperature.

Techniques: RNA Sequencing, Control, Fluorescence, Transduction, BrdU Incorporation Assay, Labeling, Construct, Negative Control, Cell Culture