Fig. 4
uPARAP downregulation impairs LEC chemotactic migration toward a VEGF-C gradient. LECs were transfected with a siRNA targeting uPARAP (siU1 and siU2) or a control siRNA (Ctr). RT-PCR (a) and Western blot (b) analyses of uPARAP, VEGFR-3 and VEGFR-2 expression (28S and GAPDH = internal controls). c Proliferation upon VEGF-C or VEGF-A stimulation. Data are those of one representative assay out of three (n = 12 for T0 and n = 8 for T48 h, biological replicates). d Scratch assay in the presence of control medium (n = 10), VEGF-C (n = 10) or VEGF-A (n = 3) (biological replicates in all conditions). e Phalloidin and uPARAP stainings of LECs migrating toward a VEGF-C or VEGF-A gradient in Ibidi µ-slides. Bars = 25 µm in left images and 50 µm in right images (higher magnification of inserts). f Boyden Chamber assay in the presence of control medium, wild type VEGF-C, VEGF-A, or mutated VEGF-CCys156Ser. Histograms correspond to the mean (n = 6 for control medium, VEGF-C and VEGF-A, and n = 3 for mutated VEGF-CCys156Ser, biological replicates) ± SEM. g Chemotactic response to a gradient of complete medium, VEGF-C or VEGF-A. The rose diagram represents the direction of migration of more than 30 cells. Black arrows indicate the mean migration direction. The absence of arrow in VEGF-C diagrams reflects impaired directionality upon uPARAP silencing. Histograms represent the migratory speed (µm/min) where results are expressed as mean (n = 6 biological replicates for complete medium and VEGF-C; n = 3 biological replicates for VEGF-A) ± SEM. Statistical analyses were performed using a non-parametric Mann–Whitney test. *P < 0.05 and **P < 0.01
