Figure 3. Forkhead box O1 (FOXO1) plays a major role in tumor necrosis factor-alpha (TNF-α)- or advanced glycation endproduct (AGE)-induced apoptosis in retinal pericytes. A: Primary cultures of retinal pericytes were transfected with different small interfering RNAs (siRNAs) for 48 h. Cells were then stimulated with TNF-α (20 ng/ml) for 1 h. After nuclear extraction, activation of FOXO1 was measured by electrophoretic mobility shift assay (EMSA). Unlabeled FOXO1 in excess was used as a competitive inhibitor. A probe with nonspecific sequence (nonspecific oligonucleotide) was used as a negative control. The experiment was performed three times with similar results. B and C: Primary cultures of retinal pericytes were transfected with different siRNAs for 48 h. Cells were then stimulated by TNF-α (20 ng/ml B) or carboxymethyllysine (CML)-collagen (200 μg/ml C) for 24 h. Apoptosis was determined by ELISA. Each value represents the mean of three replicates±standard error of the mean and is representative of three experiments. *, p<0.05 significantly differs from control; **, p<0.05, significantly differs from TNF-α- or CML-collagen-stimulated cells.