Caspase-3 activation and apoptosis upon xanthurenic acid (XAN) accumulation in vascular smooth muscle cells (VSMC). a, VSMC after one week of growth in MEM without XAN (left panel), and in the presence of XAN at a concentration of 5 μg/ml (right panel). The nuclear DNA fragmentation at 40 μg/ml, see Fig. 2b. b, Concentration of xanthurenic acid in the cell extract used for Western blot analysis. XAN was determined in the cell extracts as described previously . c, Apoptotic cell death measured as a ratio of the cells with condensed or fragmented nuclei to total nuclei. d, Caspase-3 activity measured by caspase-3 substrate Ac-DEVD-pNa cleavage, e, Western blot of active caspase-3 in VSMC after exposure to XAN. Procaspase-3 (CPP32) was cleaved in the presence of XAN with the formation of pl7 and pl2. f, Immunofluorescence staining for caspase 3 pl7 in control cells (left panel) and in VSMC exposed to 40 μg/ml of XAN for one week (right panel).