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Published in final edited form as: Free Radic Biol Med. 2012 Apr 19;53(1):109–121. doi: 10.1016/j.freeradbiomed.2012.03.023

Fig. 6 — 8-OHdG inhibits Rac1 activity. (A, B) Western blot analysis of Rac1-GTP and its downstream JNK phosphorylation. Values are means±SE of 3–4 experiments. *P<0.05 vs. control, ^†P<0.05 vs. Ang II (1 μm) treated cells. (C) Western blot analysis of VSMCs transfected with myc-tagged wild type Rac1 (Rac1-WT) or constitutively active Rac1 (Rac1-V12). Transfected cells were pretreated with 8-OHdG (100 μg/mL) for 1 h and then stimulated with Ang II (100 nM) for 24 h. Fibronectin was measured in the media collected. Anti-myc was used to monitor the transfection efficiency. (D) Real-time PCR results of ICAM-1, VCAM-1, Nox-1, and fibronectin in transfected cells. Transfected cells were pretreated with 8-OHdG (100 μg/mL) for 1 h and then stimulated with PDGF-BB (10 ng/mL) for 24 h. *P<0.05 vs. Rac1-WT, ^†P<0.05 vs. PDGF-BB treated Rac1-WT, ^‡P<0.05 vs. PDGF-BB and 8-OHdG treated Rac1-WT. (E) Scheme for Rac1 regulation. Rac1 interacts with GDP and GTP (in orange) via their base (B) and phosphate moieties (PP for GDP and PPP for GTP). 8-OHdG (in magenta) could bind to the Rac1 GEF complex, stabilize the ternary complex of Rac1 8-OHdG GEF, and stop the activation of Rac1. The loose and tight interactions of Rac1 with the nucleotides and GEF are marked in subscript L and T, respectively. (F) Docking study showing 8-OHdG binding well at the base-binding site of Rac1 GEF, and the enlarged view of the detailed interactions. 8-OHdG and 8-oxodG are depicted in capped-stick with their carbon color in magenta and purple, respectively, and the interacting residues are in thin-stick with their carbon color in green. The hydrogen bonds are marked in black dashed lines. Rac1 structures bound with GDP_(Mg), GTP_(Mg), and GEF are represented in their secondary structures and colored in brown, sky-blue, and green, respectively. GEF is displayed in gray ribbons. The ligands are depicted in spacefill with their carbon color in white for GDP and GTP, and magenta for 8-OHdG. The non-polar hydrogens are undisplayed for clarity. GEF; guanosine exchange factor, GAP; GTPase-activating protein.

Fig. 6 — 8-OHdG inhibits Rac1 activity. (A, B) Western blot analysis of Rac1-GTP and its downstream JNK phosphorylation. Values are means±SE of 3–4 experiments. *P<0.05 vs. control, ^†P<0.05 vs. Ang II (1 μm) treated cells. (C) Western blot analysis of VSMCs transfected with myc-tagged wild type Rac1 (Rac1-WT) or constitutively active Rac1 (Rac1-V12). Transfected cells were pretreated with 8-OHdG (100 μg/mL) for 1 h and then stimulated with Ang II (100 nM) for 24 h. Fibronectin was measured in the media collected. Anti-myc was used to monitor the transfection efficiency. (D) Real-time PCR results of ICAM-1, VCAM-1, Nox-1, and fibronectin in transfected cells. Transfected cells were pretreated with 8-OHdG (100 μg/mL) for 1 h and then stimulated with PDGF-BB (10 ng/mL) for 24 h. *P<0.05 vs. Rac1-WT, ^†P<0.05 vs. PDGF-BB treated Rac1-WT, ^‡P<0.05 vs. PDGF-BB and 8-OHdG treated Rac1-WT. (E) Scheme for Rac1 regulation. Rac1 interacts with GDP and GTP (in orange) via their base (B) and phosphate moieties (PP for GDP and PPP for GTP). 8-OHdG (in magenta) could bind to the Rac1 GEF complex, stabilize the ternary complex of Rac1 8-OHdG GEF, and stop the activation of Rac1. The loose and tight interactions of Rac1 with the nucleotides and GEF are marked in subscript L and T, respectively. (F) Docking study showing 8-OHdG binding well at the base-binding site of Rac1 GEF, and the enlarged view of the detailed interactions. 8-OHdG and 8-oxodG are depicted in capped-stick with their carbon color in magenta and purple, respectively, and the interacting residues are in thin-stick with their carbon color in green. The hydrogen bonds are marked in black dashed lines. Rac1 structures bound with GDP_(Mg), GTP_(Mg), and GEF are represented in their secondary structures and colored in brown, sky-blue, and green, respectively. GEF is displayed in gray ribbons. The ligands are depicted in spacefill with their carbon color in white for GDP and GTP, and magenta for 8-OHdG. The non-polar hydrogens are undisplayed for clarity. GEF; guanosine exchange factor, GAP; GTPase-activating protein.