Silencing of S1PL increases intracellular S1P content in HPAECs and stimulates cell migration in an <i>in vitro</i> scratch and in a wound healing ECIS assay.

<p>HPAECs (∼50% confluence) grown on 35-mm dishes or on gold electrodes were transfected with either scrambled siRNA or S1PL siRNA (50 nM, 72 h), then starved for 3 h in 0.1% FBS in EBM-2 without growth factors. <b><i>A</i></b> - Cell lysates (20 µg total proteins) were subjected to SDS-PAGE and Western blotted with anti-S1PL antibody as described under “Experimental procedures”. Western blot is representative of three independent experiments. <b><i>B,C</i></b> – S1PL was silenced with siRNA (50 nM, 72 h) then intracellular <i>(</i><b><i>B</i></b><i>)</i> and extracellular <i>(</i><b><i>C</i></b><i>)</i> S1P content was determined by LC/MS/MS. Ortho-vanadate (1 mM) was applied 30 min before lipid extraction. S1P level in the medium was normalized per cellular phospholipid content. <b><i>D</i></b> – HPAECs (∼50% confluence) were transfected with either scrambled siRNA or S1PL siRNA (50 nM, 72 h) then wounded on the gold electrodes as described under “Experimental Procedures” prior to S1P (1.0 µM) challenge. Transendothelial electrical resistance (TER) was recorded using an electrical cell substrate impedance-sensing system (ECIS) for 16 h. Values are the mean ± S.E.M. for three independent experiments in triplicate. <b><i>E -</i></b> HPAECs (∼50% confluence) were transfected with either scrambled siRNA or S1PL siRNA (50 nM, 72 h) prior to scratching the cells for migration assay. Scratched cells were challenged with S1P (1.0 µM) for 16 h. The closure of the wound was evaluated as described under “Experimental Section” 16 h after the wounding of EC monolayer. The values are mean ± S.E.M. for three independent experiments in triplicates.