S1P-induced in vitro lymphangiogenesis was mediated through the S1P1/Gi protein. (A) Total RNA (100 ng) from HUVECs (□) or HLECs (■) was amplified using primers for S1P receptors and β-actin. For quantification, the targets were normalized to β-actin as an internal standard. (B) After a 2-hour preincubation with 100 ng/mL PTX, HLECs were treated with 100 ng/mL S1P and 100 ng/mL PTX for an additional 4 hours. The migrated HLECs were stained and counted in 3 random fields. (C) HLECs were laid on a GFR Matrigel-coated 24-well plate and incubated with 100 ng/mL S1P and 100 ng/mL PTX for 6 hours. Two randomly chosen fields per well were photographed and the total tube area was analyzed using Scion Image. All values are expressed as means (± SD). Data are representative of 3 independent experiments with similar results. NS and ** indicate no significant difference and a statistically significant difference (P < .01), respectively.
