Fig. 2. TRIOL activates RBC-NOS through PI3P kinase-Akt signaling axis. Effect of NOS inhibitor L-NAME or activator L-arginine on the oxysterol-induced RONS formation (A) and PS-externalization (B). Arithmetic means±SD (n=6) of DCF-associated MFI (mean fluorescence intensity) and AnnexinV-FITC associated cell fluorescence measured after a 2 h or 24 h incubation, respectively, incubation of RBCs with oxysterol, preceded by 1 h pre-treatment in the presence of inhibitor, stimulant or vehicle. (C) DAF-FM associated fluorescence in RBCs treated with oxysterols. RBCs were loaded with DAF-FM diacetate and incubated for 2 h in the absence (control) or in the presence of oxysterols. Imagine representative of three separate experiments carried out in duplicate with comparable results. (D) Western blot analysis with densitometric analysis of proteins from RBCs incubated for 1h with vehicle (control) or oxysterols (columns 2,4) or pre-treated for 1 h with LY294002 before 1 h incubation with TRIOL (column 3). b-actin was used as the internal control. Imagine representative of four separate experiments. *Significantly different vs control (P<0.0001); § significantly different vs samples of the relevant group pre-incubated in the absence of inhibitor (P<0.0001) (one-way Anova associated with Bonferroni's post test).