ROS levels in MnSOD-RNAi–transfected and catalase- and glutathione peroxidase–inhibited cells. (A) EPCs were transfected with MnSOD-RNA-interference (RNAi) or scrambled RNAi, both labeled with hexachlorofluorescein (HEX). After 36 hours, HEX-positive EPCs (highly positive EPCs in gate R1) were sorted by FACS. Then HEX-positive EPCs were lysed and expression of MnSOD was measured by Western blot. Tubulin served as loading control. A representative blot is shown. (B) EPCs were incubated with AT (500 μM) and BSO (10 μM) 19 hours before FACS analysis or transfected with MnSOD-RNAi 36 hours before FACS analysis. To measure CM-DCF fluorescence in transfected cells, only the HEX-positive EPCs were subjected to further analysis of CM-DCF by selective gating. Data are mean ± SD, n = 3. *P < .05 versus MnSOD-RNAi and versus scrambled RNAi with AT and BSO. (C-D) Combination of MnSOD-RNAi transfection, AT, and BSO incubation. (E) EPCs were transfected with MnSOD-RNAi or scrambled RNAi, both labeled with hexachlorofluorescein (HEX). After 24 hours, cells were treated with H₂O₂ (500 μM) for another 24 hours. Then migration of surviving HEX-positive (trypan blue–negative) EPCs was analyzed using VEGF as stimulus in a modified Boyden chamber. Data are mean ± SD (% of scrambled), n = 3. *P < .05 versus scrambled.