S1P-induced lymphangiogenesis was mediated through the S1P1. (A) HLECs were infected with retroviruses carrying S1P1 and S1P3 shRNA expression vectors in pRS plasmid, after which stably transfected cells were obtained by selection with puromycin. pRS plasmid was used as control. After a 4-hour incubation with 100 ng/mL S1P, migrated HLECs were stained and counted in 3 random fields (□). HLECs were laid on a GFR Matrigel-coated 24-well plate and incubated with 100 ng/mL S1P for 6 hours. Two randomly chosen fields per well were photographed and the total tube area was analyzed using Scion Image (■). (B,C) Effect of S1P1 antagonist or S1P1 control molecule on the S1P-induced migration (B) and capillary-like tube formation (C) of HLECs was examined as described in panel A. (D,E) Ten days after subcutaneous injection of Matrigel in C57BL/6 mice (5 mice per group), the Matrigel was removed, fixed, embedded in paraffin, sectioned at 4 μm, and immunostained using antibodies specific for Prox-1 (green) and podoplanin (red). (D) Representative photographs of untreated control mice, and mice treated with S1P (0.4 μg) in the absence or presence of S1P1 antagonist (100 μM) or S1P1 control molecule (100 μM). Scale bars represent 50 μm. (E) The number of Prox-1⁺/podoplanin⁺ lymphatic vessels per field was counted. All values are expressed as means plus or minus SD (A-C) and means plus or minus SEM (E). In panels A-C, data are representative of 3 independent experiments with similar results. ** indicates a statistically significant difference (P < .01).