Effect of StSPL on S1P-stimulated MAPK phosphorylation, cell proliferation and migration of endothelial cells.

<p>(A) Quiescent EA.hy 926 human endothelial cells were treated for 10 min with either vehicle (Co) or S1P (1 µM) in the absence or presence of WT StSPL (StSPL; 10 µg/ml) or the K311A mutant (10 µg/ml). Cell lysates were prepared and separated by SDS-PAGE, transferred to nitrocellulose and subjected to Western blotting using antibodies against phospho-p42/p44 (dilution of 1∶1000, upper panel) and total p42/p44-MAPK (dilution each 1∶6000, lower panel). Data are representative of four independent experiments. (B) Quiescent cells were treated for 28 h with either vehicle (-) or S1P (1 µM), which had been pretreated for 30 min at 37°C with either vehicle (-), WT StSPL (StSPL) or the K311A mutant, in the presence of [³H]thymidine. Incorporated radioactivity was measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022436#s4" target="_blank">Materials and Methods</a> section. Results are expressed as cpm/well of incorporated [³H]thymidine and are means ±S.D. (n = 4). ***p<0.001 considered statistically significant when compared to the vehicle treated control values; ^##p<0.01, ^###p<0.001 when compared to the S1P-treated values by one-way ANOVA analysis and Bonferroni post test. (C) Quiescent cells were treated for 14 h with DMEM (Co) or S1P (1 µM) which had been pretreated for 30 min at 37°C with either vehicle (-), WT StSPL (StSPL) or the K311A mutant. Thereafter, migrated cells were analysed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022436#s4" target="_blank">Materials and Methods</a> section. Results are expressed as migrated cells per counted field and are means ±S.D. (n = 3). ***p<0.001 considered statistically significant when compared to the vehicle treated control values; ^###p<0.001 when compared to the S1P-treated values.