DAPI solution, respectively, in dark chamber. After washing with 0.05% Tween 20-PBS, the specimens were mounted in Fluor- omount Aqueous mounting medium (Sigma). Confocal laser scanning microscopy was carried out with a Leica TCS SP5 confocal microscope (Wetzlar, Germany) using an oil immersion objective. Pull-down assay: measurement of Rho GTPases activity-The ability of Rac1-GTP and Cdc42-GTP to bind GST-PAK1-PBD (p21-activated kinase-binding domain) and the ability of RhoA- GTP to bind to GST-Rhotekin-RBD (Rho binding domain) were used to determine the cellular levels of active GTPases. Small Rho GTPases GTP pull-down assays were performed by incubating the cleared lysates with glutathione agarose beads that were coupled to GST-PAK1-PBD or GST-Rhotekin-RBD (Millipore) for 1 h at 4uC. The beads were then followed by four washes in washing buffer. Bound proteins were solubilized by the addition of 25 ml of SDS-PAGE Laemmli loading buffer and separated by 12% SDS- PAGE, followed by western blotting for Rac1, Cdc42 or RhoA proteins. G-LISA (ELISA-based GTPase activation assays): measurement of the GTP-bound form of Rac1-After protein lysates being collected, the activated GTP-bound Rac1 was analyzed with a G- LISA activation assay biochemistry kit (Cytoskeleton, Denver, CO, USA) according to the manufacturer’s instructions. In vitro reaction assay between Rac1 and H2S-To detect the direct action of H2S on Rac1, we used an in vitro reaction system. GTP (200 mM) and different concentrations of NaHS were respectively added into 50 ml reaction buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1% lgepal CA-630, 10 mM MgCl2, 10 mM EDTA and 2% glycerol) that contained 20 ng human recombinant Rac1 (Cytoskeleton). We used GDP (200 mM) instead of GTP as a negative control and GTPcS (200 mM) instead of GTP as a positive control separately in the reaction system. After incubation at 30uC for 30 min, the reaction was stopped, and the activated form of Rac1 was assayed using a G- LISA assay kit (Cytoskeleton) in accordance with the manufac- turer’s instructions. Statistical analysis-Quantitative data are presented as the means 6 SE. Differences between groups were analyzed using one-way ANOVA followed by post-hoc Tukey’s test, where applicable. In all cases, a P value of ,0.05 was taken to indicate statistical significance. Results H2S promotes cell migration and microvessel tube formation in human endothelial cells-To test the action of H2S on HUVECs, we used sodium hydrogen sulfide (NaHS) as a precursor for H2S. Exposure of HUVECs to 50 mM NaHS promoted an increase in the cell migration rate with an approximate 2-fold increase in both the scratch wounding assay (NaHS vs. Vehicle: 197.8624.4 mm vs. 96.6617.5 mm; P,0.05; Fig. 1A, B) and the transwell boyden chamber assay (NaHS vs. Vehicle: 121614 vs. 62614; P,0.05; Fig. 1C, D). Furthermore, 50 mM NaHS significantly enhanced capillary-like structure formation of HUVECs cultured on re- duced-growth factor matrigel, which was reflected by both of tube length (NaHS vs. Vehicle: 6902.66717.2 mm vs. 4592.76567.2 mm; P,0.05; Fig. 1E, F) and number of tube branches (NaHS vs. Vehicle: 21.963.9 vs. 10.362.3; P,0.05; Fig. 1E, G). H2S induces actin cytoskeleton reorganization in endothelial cells-As shown with immunostaining of F-actin (Fig. 2A), an endothelial cell without NaHS treatment appeared spindle- shaped with regular paralleled actin filaments across the cell H2S Signals through VEGFR-PI3K-Rac1-Cofilin body. The stress fibers were distributed evenly in the cortical region of the cell. In contrast, in cells treated with NaHS, there were significant changes regarding the cellular actin cytoskeleton. We observed that after 5 min exposure to 50 mM NaHS, the endothelial cell formed protrusions in the lateral body of the cell (depicted by an arrowhead in Fig. 2A). Following prolonged NaHS treatment, the typical lamellipodia were generated most prominently in the leading cell edge (depicted by a thin arrow in Fig. 2A) and the characteristically shaped membrane ruffles were apparent. As shown in Fig. 2A, the NaHS-induced augmentation of typical lamellipodia in the leading cell edge indicates the reorganization of the actin cytoskeleton. As depicted in Fig. 2A, paxillin is specifically localized to focal adhesions, which are used to anchor stress fibers (depicted by the thick arrow in Fig. 2A). Compared with control cells, treatment with NaHS did not change the localization of paxillin. The paxillin-containing focal adhesion had no evident turnover transmission or redistribution after NaHS treatment. In addition, the phosphorylation of paxillin on Ser126 and Tyr118 did not change (Fig. 2B). H2S selectively activates the Rho GTPase Rac1 in HUVECs- The results from pull-down assays demonstrated that Rac1 was quickly activated after treatment with NaHS for 10 min (Fig. 3A), whereas RhoA and Cdc42 were not activated by NaHS (Fig. 3A, B). To verify that Rac1 was activated by NaHS, we further used a G-LISA assay. The data indicate that Rac1 was activated by 50 mM NaHS in a time-dependent manner and reached a peak at 12 min (Fig. 3C). It should be noted that the activation of Rac1 was prompt and temporary; a three- min treatment with NaHS induced activation of Rac1, which declined rapidly to normal after 30 min of treatment. H2S does not directly activate Rac1 in vitro-The purified recombinant Rac1 was allowed to react with different concentra- tions of NaHS varying from 0–200 mM. G-LISA assay was conducted to detect the activity of Rac1 after each reaction. The results showed no significant difference between groups (Fig. 3D), revealing that H2S did not interact with Rac1 in the in vitro cell- free system. Together, these results suggest that Rac1 may be activated by an upstream molecule that is directly activated by H2S, which in turn stimulates Rac1. Rac1 mediates H2S-induced lamellipodia formation-The EGFP-Rac1-fusion protein was expressed in HUVECs 48 hours after transfection with EGFP-Rac1-T17N but was not expressed in cells that were transfected with the EGFP control vector (Fig. 4A). Examination of the actin cytoskeleton indicated that the H2S- mediated induction of the typical wavy lamellipodia (indicated by a thin arrow in Fig. 4B) at the cell edge was inhibited by the presence of dominant negative Rac1. Moreover, fragments of disrupted actin filaments (indicated by a thick arrow in Fig. 4B) in the cell body were observed in cells that were transfected with dominant negative Rac1. H2S-induced endothelial cell migration and microvessel tube formation are both dependent on Rac1-Transfection of Rac1 siRNA and GAPDH siRNA (positive control) specifically knocked down the expression of their respective target genes (Fig. 4C) and proteins (Fig. 4D). As shown in Fig. 5, compared to the EGFP control vector group, dominant negative Rac1 significantly blunted the cell migration effect caused by the 50 mM NaHS treatment both in the scratch wounding assay (Fig. 5A, B) and the transwell boyden chamber assay (Fig. 5C, D). Similarly, Rac1 siRNA induced a marked reduction in the promoted migration rate that was induced by NaHS (Fig. 5A, B, C, D). In the tube formation assay, both the dominant negative Rac1 and Rac1 PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e44590