Dominant negative Rac1 inhibits the H₂S-induced reorganization of the actin cytoskeleton in HUVECs.

<p>(A) HUVECs were transfected with plasmids encoding EGFP only (EGFP) or dominant-negative Rac1 (EGFP-Rac1-T17N). Western blot analysis showed manifest levels of the EGFP-Rac1 fusion protein in HUVECs transfected with EGFP-Rac1-T17N vector but not in the cells transfected with EGFP control vector. (B) Transfected cells were starved and then treated with or without 50 µM NaHS for 12 min. Cells were then fixed and stained with rhodamine-labeled phalloidin for F-actin and anti-EGFP antibody for EGFP-tagged Rac1. Representative images are shown. Thin arrows point to the formed lamellipodia. Thick arrows indicate the fragments of disrupted F-actin around the nucleus. Scale bars, 10 µm. (C, D) Rac1 siRNA decreased the expression of Rac1 both on the mRNA levels (C) and protein levels (D). (C) Electroporation was used to transfect HUVECs with siRNA. 48 h after transfection, mRNA transcripts of Rac1 and GAPDH as measured by real-time PCR were significantly reduced by siRNA separately. (D) Representative blots were shown on the protein levels after 72 h transfection. Transfection of HUVECs with Rac1 siRNA and GAPDH siRNA specifically knocked down the expression of their respective target genes without affecting each other’s targets. Values represent the means ± SE, <i>n</i> = 9. *<i>P</i><0.05.