S1P induced migration and the formation of capillary-like tube structure of human lymphatic endothelial cells. (A) After 4 hours of incubation with S1P or VEGF-C, migrated HLECs were stained and counted in 3 random fields. (B) S1P or VEGF-C was added to serum-starved HLECs for 24 hours, followed by additional incubation for 12 hours with 0.5 μCi (0.0185 MBq) [³H]-thymidine in HE-SFM. Results are expressed as the percentage [³H]-thymidine incorporation of the control versus S1P- or VEGF-C–treated HLECs. (C) HLECs were laid on a 24-well, GFR Matrigel-coated plate and incubated with S1P or VEGF-C for 6 hours. Two randomly chosen fields per well were photographed and the total tube area was analyzed using Scion Image. (D,E) Effect of S1P on the migration (D) and capillary-like tube formation (E) of HMVECs-dLy, lymphatic endothelial cells derived from dermal skins, was examined as described in panels A and C, respectively. (F) Cultured HLECs and HMVECs-dLy were fixed, and immunostained using antibodies against Prox-1 (green), LYVE-1 (red), and podoplanin (green). The nuclei were counterstained by Hoechst (blue). Scale bars represent 50 μm. Note that HLECs and HMVECs-dLy were not immunolabeled by isotype-matched control IgG antibodies (data not shown). All values are expressed as means (± SD). Data are representative of 3 independent experiments with similar results. * and ** indicate statistically significant differences (P < .05 and P < .01, respectively).