HKa causes consumption of cellular GSH and phospholipid peroxidation in endothelial cells. (A) To assess GSH consumption, endothelial cells were cultured in 10 cm² gelatin-coated dishes at a concentration of 3 × 10⁴ cells/mL and stimulated with 10 ng/mL bFGF in the absence or presence of 50 nM HKa. At various time points, cell lysates were prepared as described under “Determination of cellular GSH content” in “Materials and methods” and, after precipitation of cellular proteins, were analyzed for GSH content by measuring the fluorescence of added o-phthaldialdehyde (24 mM final concentration). Residual protein content was measured in parallel, and the concentration of intracellular GSH was expressed as nanomoles per mg of protein. (B) To assess lipid peroxidation, cells were cultured as described for panel A and stimulated with bFGF in the absence or presence of 50 nM HKa. Lysates were prepared as in panel A, and total MDA and 4-HNE content was determined using a commercial Lipid Peroxidation Assay Kit, as described in “Materials and methods.” Protein content was measured in parallel, and the concentration of lipid peroxides was expressed as nanomoles per mg protein. Error bars indicate standard deviation of triplicate points.