H2S Signals through VEGFR-PI3K-Rac1-Cofilin Figure 1. H2S promotes endothelial cell migration and microvessel tube formation. (A) Representative micrographs of monolayer wounding assay in HUVECs treated with or without NaHS (50 mM) at 0 h and 6 h after treatment. (B) Statistical analysis of the scratch wounding assay. (C) Cell migration was also assessed by transwell boyden chamber assay. Shown are the representative micrographs and the values (D) of the migrated cells treated with 50 mM NaHS. (E) Representative micrographs of microvessel tube formation in HUVECs treated or not treated with NaHS (50 mM). Statistical analysis of tube length (F) and branching points (G). Data represent the means 6 SE of five independent experiments. Each experiment was performed in duplicate. *P,0.05. doi:10.1371/journal.pone.0044590.g001 siRNA significantly blunted vessel-like structure formation after treatment with NaHS (Fig. 5E, F, G). H2S signals through the VEGFR-PI3K pathway to mediate the activation of Rac1 and to promote cell migration in HUVECs- HUVECs that were exposed to 50 mM NaHS exhibited a sustained increase in Akt phosphorylation that was evident as early as 5 min and that lasted up to 2 h (Fig. 6A, B) post treatment. Moreover, treatment with 50 mM NaHS led to ERK (Fig. 6A, B) and cofilin phosphorylation (Fig. 6A, B), albeit with different kinetics, e.g., phosphorylation of ERK was rapid and transient, whereas cofilin phosphorylation showed a delayed but more sustained pattern. With the exception of ERK, there was no obvious change in the PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e44590