Figure 4.

TSP1 (thrombospondin-1)-induced CD47 (cluster of differentiation) activation blocks VEGF (vascular endothelial growth factor)-C–stimulated lymphangiogenic signaling. A through D, Lymphatic endothelial cells (LECs) were pretreated with TSP1 (22 nM, 16 h) in 0.5% fetal bovine serum (FBS) containing basal media MV2, stimulated with VEGF-C (15 min), and cell lysates subjected to Western blot analysis. A, Representative Western blot images are shown. B through D, Bar diagrams represent mean protein levels expressed as a ratio of phospho-to-total proteins, AKT (B), eNOS (C), and ERK1/2 (D; n=3). E, LECs were treated with control or CD47-siRNA (48 h) and immunoblotting done to determine CD47 expression (ab175388). F through I, Control or CD47-siRNA–treated cells were treated as in Figure 4A and Western blot experiments executed. F, Representative Western blot images are shown. G through I, Bar diagrams represent pAKT/total AKT (G), peNOS/total eNOS (H), and pERK1/2/total ERK1/2 (I; n=3). J, Control or CD47-silenced LECs were pretreated with TSP1 (22 nM, 4 h), stimulated with VEGF-C (100 ng/mL, 1 h) and analyzed for NO (nitric oxide) production using DAF-FM diacetate. K, Control or CD47-silenced LECs were pretreated with vehicle or TSP1 (1 h), incubated with H2DCFDA solution, and fluorescence analyzed using flow cytometry. Statistical analyses were performed using 1-way ANOVA (B–D), 2-way ANOVA with Tukey test for multiple comparisons (G–I and K), and 2-tailed unpaired Student t test (J). Data represent mean±SEM.