αHS-induced proliferation of ECs and transformed cells depends on p38 MAPK signalling activation. A,

<p>HUVECs were incubated in serum free medium with or without αHS (AO4B08; 1.5 µg/ml) for 5, 15, 45, and 60 min, and phosphorylated receptor tyrosine kinase proteins were determined on pooled cell lysates by human anti-phospho receptor tyrosine kinase antibody array analysis as described under <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049092#s4" target="_blank">Methods</a></i>. Shown are representative immunoblots from two independent experiments. Indicated areas show FGFR, column 1: FGFR1; 2: FGFR2; 3: FGFR3; 4: FGFR4. PDGFR, column 1: PDGFRα; 2: PDGFRβ; 3: SCFR; 4: Flt3. VEGFR, column 1: VEGFR1; 2: VEGFR2; 3: VEGFR3. <b>B</b>, HUVECs were incubated in serum free medium with or without αHS (EV3C3; 1.5 µg/ml) for 5, 15, 45, and 60 min, and phosphorylated kinase proteins were determined on pooled cell lysates by human anti-phospho kinase antibody array analysis as described under <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049092#s4" target="_blank">Methods</a></i>. Left panels show representative immunoblots from three independent experiments. Right panel shows quantification of p-p38 MAPK, p-CREB, and p-ERK1/2 levels at the various conditions, presented as relative intensities <i>vs.</i> array reference (red box). <b>C</b>, HUVECs were either untreated (Ctrl) or incubated with αHS (AO4B08;1.25 µg/ml) for the indicated time periods followed by immunoblotting for phospho-p38 MAPK, total p38 MAPK and α-tubulin. Left panel shows representative immunoblots of three separate experiments. Right panel shows quantification of relative phospho-p38 MAPK levels (P-p38/α-tubulin) from a representative experiment. <b>D</b>, Same experiment as in (C) was performed with mouse lung microvascular ECs, showing comparable induction of p38 MAPK phosphorylation by αHS as in HUVECs. <b>E–F</b>, αHS-induced proliferation depends on p38 MAPK. Proliferation was determined by [³H]thymidine incorporation in mouse ECs (<b>E</b>) and U-87 MG glioblastoma cells (<b>F</b>) following treatment with the p38 MAPK inhibitor SB203580 (1 µM) and/or αHS (AO4B08; 1.25 µg/ml) for 3 h, as indicated. Basal cell proliferation was not significantly affected by p38 MAPK inhibition whereas αHS induced proliferation was almost completely reversed. * Statistically different from untreated control, P<0.05; # Statistically different from αHS-treated, P<0.05.