Journal: Scientific Reports

Article Title: PSGL-1 decorated with sialyl Lewis a/x promotes high affinity binding of myeloma cells to P-selectin but is dispensable for E-selectin engagement

doi: 10.1038/s41598-024-52212-2

Figure Lengend Snippet: PSGL-1 is a carrier for SLe a/x -related structures in the MM1S Heca452 cells. Membrane-enriched fractions from the MM1S Heca452 and the MM1S Parental cells were subjected to an immunoprecipitation using the Heca452 ( A , B ) or PSGL-1 ( C , D ) antibodies and their matched isotype controls. The eluted protein complexes were run on an SDS-PAGE, transferred to a nitrocellulose membrane and blotted for Heca452 ( A , D ) or PSGL-1 ( B , C ). Labels above the blots represent the different samples. Numbers on the left hand side represent the molecular weight marker. Western blot analysis depicted in the figure is representative of 2 independent experiments.

Article Snippet: In some experiments, cells were pre-incubated for 1 h before the rolling assay with a mouse anti-human PSGL-1 blocking antibody (clone 688,102; Bio-techne) or mouse isotype control (clone 11711; Bio-techne) at 10 µg/ml or pre-treated/mock-treated with neuraminidase from Vibrio cholerae (1 mU/ml) or pronase (1 mg/ml) for 45 min at RT in serum free RPMI1640 media.

Techniques: Membrane, Immunoprecipitation, SDS Page, Molecular Weight, Marker, Western Blot

Journal: Scientific Reports

Article Title: PSGL-1 decorated with sialyl Lewis a/x promotes high affinity binding of myeloma cells to P-selectin but is dispensable for E-selectin engagement

doi: 10.1038/s41598-024-52212-2

Figure Lengend Snippet: Downregulation of PSGL-1 inhibits rolling and adhesion on P-selectin but not on E-selectin. MM1S Heca452 cells stably expressing a scramble shRNA control or the shRNA for PSGL-1 were tested in a rolling and adhesion assay under shear stress on recombinant E-selectin ( A ) and P-selectin ( B ), both at 15 µg/ml. Histograms represent the mean + sem of three independent experiments performed in duplicate. Symbols on top of the error bars represent the statistical significance of the rolling (white) or adhesion (grey) fractions compared to the same fractions in the scramble control. Symbols on top of the horizontal bars represent the statistical significance of the total fraction (rolling plus adhesion fraction). The unpaired one-tailed t test was used to determine statistical significance. **p < 0.01; *p < 0.05; ns non-significant.

Article Snippet: In some experiments, cells were pre-incubated for 1 h before the rolling assay with a mouse anti-human PSGL-1 blocking antibody (clone 688,102; Bio-techne) or mouse isotype control (clone 11711; Bio-techne) at 10 µg/ml or pre-treated/mock-treated with neuraminidase from Vibrio cholerae (1 mU/ml) or pronase (1 mg/ml) for 45 min at RT in serum free RPMI1640 media.

Techniques: Stable Transfection, Expressing, shRNA, Cell Adhesion Assay, Shear, Recombinant, One-tailed Test

Journal: Scientific Reports

Article Title: PSGL-1 decorated with sialyl Lewis a/x promotes high affinity binding of myeloma cells to P-selectin but is dispensable for E-selectin engagement

doi: 10.1038/s41598-024-52212-2

Figure Lengend Snippet: PSGL-1 neutralization with a blocking antibody inhibits rolling and adhesion on P-selectin but not on E-selectin. MM1S Heca452 ( A , C ) and RPMI8226 Heca452 ( B , D ) cells were pre-incubated for 1 h with 10 µg/ml of an anti-PSGL-1 blocking antibody or matched-isotype control and then tested in a rolling and adhesion assay under shear stress on recombinant E-selectin ( A , B ) and P-selectin ( C , D ), both at 15 µg/ml. Histograms represent the mean + sem of three independent experiments performed in duplicate. Symbols on top of the error bars represent the statistical significance of the rolling (white) or adhesion (grey) fractions compared to the same fractions in the isotype control. Symbols on top of the horizontal bars represent the statistical significance of the total fraction (rolling plus adhesion fraction). The unpaired one-tailed t test was used to determine statistical significance. ***p < 0.001; **p < 0.01; ns non-significant.

Article Snippet: In some experiments, cells were pre-incubated for 1 h before the rolling assay with a mouse anti-human PSGL-1 blocking antibody (clone 688,102; Bio-techne) or mouse isotype control (clone 11711; Bio-techne) at 10 µg/ml or pre-treated/mock-treated with neuraminidase from Vibrio cholerae (1 mU/ml) or pronase (1 mg/ml) for 45 min at RT in serum free RPMI1640 media.

Techniques: Neutralization, Blocking Assay, Incubation, Cell Adhesion Assay, Shear, Recombinant, One-tailed Test

Journal: Scientific Reports

Article Title: PSGL-1 decorated with sialyl Lewis a/x promotes high affinity binding of myeloma cells to P-selectin but is dispensable for E-selectin engagement

doi: 10.1038/s41598-024-52212-2

Figure Lengend Snippet: Neuraminidase and pronase treatments differentially affect the levels of PSGL-1 and Heca452 in the MM1S Heca452 cells. Western blot ( A , C ) and flow cytometry ( B , D ) analysis of MM1S Heca452 cells pre-treated/mock-treated with either neuraminidase (1 mU/ml) or pronase (1 mg/ml) for 45 min at RT. After treatment, cells were either lysed for Western blot analysis or stained with the indicated antibodies and analyzed by flow cytometry. Cell extracts were subjected to SDS-PAGE, transferred onto a nitrocellulose membrane and blotted for PSGL-1, Heca452 and β-actin, used as loading control. Labels above the blots represent the different treatments whereas labels on the right hand side of the blots indicate the antibodies used for blotting. Numbers on the left hand side of the blots represent the molecular weight marker. The flow cytometry analysis is reported as a band plot generated with the Infinicyt software v 2.0.5.b.007. Labels above the band plots represent the different treatments whereas labels on the left hand side of the band plots indicate the antibody used for the staining. The Western blot and flow cytometry analyses depicted in the figure are representative of 3 independent experiments.

Article Snippet: In some experiments, cells were pre-incubated for 1 h before the rolling assay with a mouse anti-human PSGL-1 blocking antibody (clone 688,102; Bio-techne) or mouse isotype control (clone 11711; Bio-techne) at 10 µg/ml or pre-treated/mock-treated with neuraminidase from Vibrio cholerae (1 mU/ml) or pronase (1 mg/ml) for 45 min at RT in serum free RPMI1640 media.

Techniques: Western Blot, Flow Cytometry, Staining, SDS Page, Membrane, Molecular Weight, Marker, Generated, Software