Tiam1 is required for flow-induced ROS production in vitro and in vivo. (A) ECs were transfected with control or Tiam1-specific siRNAs. 72 h after transfection, cells were incubated with the H2-DCFDA dye and sheared for indicated times. Shear stress was subsequently applied in continued presence of the H2-DCFDA dye for the indicated times. ROS production was assessed by measuring the fluorescence of the cell lysates at excitation 485 nm/emission 535 nm. (B) Human ECs were transfected with indicated combinations of siRNAs and rescue plasmids. 48 h after transfections, cells were lysed and lysates resolved on SDS-PAGE and analyzed by Western blotting using Tiam1-specific and actin-specific antibodies. 48 h after the transfections, cells were loaded with the H2-DCFDA dye, sheared for 30 min, or kept as static control and ROS production measured as described above. (C) Control or mouse Tiam1-specific siRNAs were introduced into C57BL/6 mice (>30 wk) through retro-orbital injection for two consecutive days. 72 h after the initial injection, the aortas were isolated and processed for en face staining. ROS production was visualized by incubating the tissue sections with H2-DCFDA dye at 37°C for 1 h. Images of aorta tissues were obtained immediately after processing the sections. Values are mean ± SEM, n = 4; *, P < 0.05; **, P < 0.01.
