S1P-induced in vitro lymphangiogenesis was mediated through the PLC/Ca²⁺ pathway. (A) After a 4-hour isncubation with 100 ng/mL S1P containing 10 μM LY294002, 1 mM L-NAME, 10 μM PD98059, 25 μM SB203580, 30 μM BAPTA-AM, or 5 μM U73122, the migrated HLECs were stained and counted in 3 random fields. (B) HLECs were laid on a GFR Matrigel-coated 24-well plate and incubated with 100 ng/mL S1P containing 10 μM LY294002, 1 mM L-NAME, 10 μM PD98059, 25 μM SB203580, 30 μM BAPTA-AM, or 5 μM U73122 for 6 hours. Two randomly chosen fields per well were photographed and the total tube area was analyzed using Scion Image. (C) HLECs were loaded with fura-2/AM for 30 minutes. The cells were resuspended in Ca²⁺-free Locke solution, transferred to a quartz cuvette, and exposed to 100 ng/mL S1P with 100 ng/mL PTX, 10 μM LY294002, 1 mM L-NAME, 10 μM PD98059, 25 μM SB203580, 30 μM BAPTA-AM, or 5 μM U73122. Relative intracellular Ca²⁺ influx was calculated from the tracing. All values are expressed as means (± SD). Data are representative of 3 independent experiments with similar results. ** indicates a statistically significant difference (P < .01).