Journal: BioMed Research International

Article Title: Inhibition of P-Selectin and PSGL-1 Using Humanized Monoclonal Antibodies Increases the Sensitivity of Multiple Myeloma Cells to Bortezomib

doi: 10.1155/2015/417586

Figure Lengend Snippet: P-selectin and PSGL-1 regulate adhesion of MM cells to endothelial and stromal cells in vitro . Endothelial and stromal cells were treated with increasing concentrations of SelG1 antibody (2.5, 5, and 10 μ g/mL) followed by adhesion of H929 or MM1.s cells labeled with calcein-AM. MM cell adhesion was assessed as a signal of adherent calcein-AM-positive MM cells measured by fluorescent reader and normalized to untreated cells (a). Likewise, H929 or MM1.s cells labeled with calcein-AM were incubated with increasing concentrations of SelK2 antibody (2.5, 5, and 10 μ g/mL) and plated on untreated endothelial and stromal cells, and the cell adhesion was measured as above (b). Stromal (c) and endothelial cells (d) were treated with SelG1 (10 μ g/mL) for 1 hr followed by plating MM1.s and H929 labeled with calcein-AM, and the adhesion was assessed as a signal of adherent calcein-AM-positive MM cells measured by fluorescent reader and normalized to untreated cells. Likewise, MM1.s and H929 were treated with SelK2 (10 μ g/mL) for 1 hr and plated on stromal (e) or endothelial cells (f), and the cell adhesion was measured as above. Values were considered significant for ∗ p < 0.05.

Article Snippet: The humanized monoclonal antibodies anti-P-selectin (SelG1) and anti-PSGL-1 (SelK2) were obtained from Selexys Pharmaceuticals (Oklahoma City, OK).

Techniques: In Vitro, Labeling, Incubation

Journal: BioMed Research International

Article Title: Inhibition of P-Selectin and PSGL-1 Using Humanized Monoclonal Antibodies Increases the Sensitivity of Multiple Myeloma Cells to Bortezomib

doi: 10.1155/2015/417586

Figure Lengend Snippet: P-selectin and PSGL-1 affect proliferation of MM cells cocultured with endothelial and stromal cells in vitro . The effect of SelG1 antibody (10 μ g/mL) used on endothelial and stromal cells and SelK2 (10 μ g/mL) used on H929 cells on MM cell proliferation, with or without bortezomib (5 nM) treatment, analyzed by flow cytometry and normalized to untreated cells. Values were considered significant for ∗ p < 0.05.

Article Snippet: The humanized monoclonal antibodies anti-P-selectin (SelG1) and anti-PSGL-1 (SelK2) were obtained from Selexys Pharmaceuticals (Oklahoma City, OK).

Techniques: In Vitro, Flow Cytometry

Journal: Cellular and Molecular Bioengineering

Article Title: Effects of Transient Exposure to High Shear on Neutrophil Rolling Behavior

doi: 10.1007/s12195-018-0533-z

Figure Lengend Snippet: Suspensions of isolated neutrophils were perfused through straight (St) and a constricted (310 msec) channel microfluidic devices, the effluents collected and labeled with an anti-PSGL-1 antibody (PL1) followed by a secondary antibody, and the expression level of PSGL-1 on the surface of neutrophils was analyzed by flow cytometry. Unsheared (Un) samples or samples incubated with 10nM PMA served as negative and positive controls. The data is shown as the mean fluorescent intensity ± standard error of the mean and is representative of five independent experiments with different donors. Statistical significance of the differences between the samples and the unsheared sample was measured using ANOVA with a Sidak post hoc test (*p<0.05).

Article Snippet: FITC tagged mouse-anti-PSGL-1 mAb PL1 was purchased from MBL (Woburn, MA), while unlabeled PL1 was purchased from Ancell (Bayport, MN).

Techniques: Isolation, Labeling, Expressing, Flow Cytometry, Incubation

Journal: Cell chemical biology

Article Title: Thioglycosides are efficient metabolic decoys of glycosylation that reduce selectin dependent leukocyte adhesion

doi: 10.1016/j.chembiol.2018.09.012

Figure Lengend Snippet: A-D. Wild-type HL-60s were cultured with 80 μM ONAP, SNAP or vehicle controls for 40 h. Flow cytometry measured cell surface carbohydrate structures using either fluorescent antibodies or lectins: PNA (binds Galβ1,3GalNAc, panel A), ECL (binds Galβ1,4GlcNAc, B), PHA-L (binds complex N-glycans, C), and CD15/Lewis-X (mAb HI98, D). Data in inset are mean fluorescence intensity ± SD for >3 independent experiments. * P<0.05 with respect to VC. SNAP increased PNA, ECL, and PHA-L bindings. ONAP has a smaller effect. E-F. Cell lysates run under standard reducing conditions, were probed with mAbs against mucinous proteins, anti-human PSGL-1 mAb TB5 (panel E) and anti-human CD43 mAb L60 (F). SNAP reduced molecular mass of both glycoproteins indicating truncation on O-glycans. G. HL-60s were treated with 80 μM glycosides or vehicle control. Cell lysates were incubated with 10,000 dpm UDP-[14C]Gal (donor) in the presence or absence of 0.5 mM GalNAc-OBn (acceptor). [14C]Gal is preferentially added to SNAP present in cell lysates rather than GalNAc-OBn (left-half). Even in the absence of GalNAc-OBn, radioactive products are formed on SNAP and ONAP (right-half). H-J. HL-60s were cultured with 80 μM ONAP, SNAP or GalNAc, with 100 μM GalNAc-OBn being added after 8h in some cases. Products secreted into culture medium over 2 days were collected and quantitatively analyzed using LC-MS/MS. SNAP, and to a lesser extent ONAP, reduces glycan biosynthesis on GalNAc-OBn (panel H). Extended carbohydrate chains grew on SNAP (panel I) and ONAP (J). All assignments were verified by MS/MS. *P<0.05, **P<0.01 with respect to GalNAc. †: No detected.

Article Snippet: Next, the proteins were transfer onto 0.2 μm nitrocellulose membrane, and probed with either anti-human PSGL-1 mAb TB5 (GeneTex, Irvine, CA) or anti-human CD43 mAb L60 (BD Biosciences).

Techniques: Cell Culture, Flow Cytometry, Fluorescence, Control, Incubation, Liquid Chromatography with Mass Spectroscopy, Glycoproteomics, Tandem Mass Spectroscopy