Fig. 3 |. Cargocytes interact with adhesion molecules.

a, Graphs show surface expression of PSGL-1 (left), P-selectin binding (middle), and E-selectin binding (right) of hT-MSCs or Cargocytes analyzed by flow cytometry. Data were analyzed in Flowjo and normalized to mode. MSC^PSGL-1, PSGL-1/Fut7 lentivirus-engineered MSC; 2hr/24hr/48hr Cargo ^PSGL-1, MSC ^PSGL-1-derived Cargocytes analyzed at indicated time point after enucleation; Parental MSC, Non-engineered MSC; Isotype control, MSC ^PSGL-1 stained with isotype matching IgG. b, Graphs show LDV-FITC binding on un-engineered Cargocytes or Cargocytes^CXCR4 analyzed by flow cytometry. Data were analyzed in Flowjo and normalized to mode. Unstained control, no LDV-FITC control; LDV-FITC, 4nM LDV-FITC; LDV-FITC+SDF-1α, 4nM LDV-FITC+500ng/ml SDF-1α; LDV-FITC+SDF-1α+LDV, 4nM LDV-FITC+500ng/ml SDF-1α+1μM LDV (unconjugated competitor). Dashed line labels the position of median fluorescence intensity. c, Bar graphs represents the MFI change of LDV-FITC binding intensity before and after SDF-1α treatment. MFI ratio = (MFI^{LDV-FITC+ SDF-1α} - MFI^{unstained control})/ (MFI^LDV-FITC - MFI^{unstained control}). n=4 biological replicates. P value, one-way ANOVA with Tukey’s multiple comparisons test. d, Representative images show the adherent MSCs or Cargocytes on control HUVECs or inflamed HUVECs (TNF-α treated). Representative images out of 24 images obtained are shown. Scale bar, 100μm. e, Bar graphs represent the adherent cell numbers per field (100X magnification). TNF-α, HUVECs pre-treated with 10ng/ml TNF-α for 6hr. SDF-1α, 500ng/ml SDF-1α; a-PSGL-1, 10μg/ml anti-PSGL-1 antibody pre-treatment; a-VLA-4, 10μg/ml anti-VLA-4 antibody pre-treatment; n= 24 random fields of 4 biological replicates. P value, one-way ANOVA with Tukey’s multiple comparisons test. All data are representative of at least 2 independent experiments.