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Figure 5: Status of p53, phospho-p53, caspase 3, cleaved-caspase 3 and apoptosis in cultured A549 cells exposed to low or high concentration of AECS/p-BQ. The figure represents immunoblots of phosphorylated p53 and p53 (a) and caspase 3 and cleaved caspase 3 (b). Cell lysate of A549 cells were either nontreated (NT) or exposed to AECS (2 μL or 50 μL/mL) or p-BQ (200 ng or 2.5 μg/mL) for 1 hr followed by incubation in serum containing media for 12 hr. Vitamin C (40 μg/mL) pretreatment of cells in serum-free media for 15 min prevented AECS/p-BQ-induced activation of p53 or cleavage of caspase 3 (a, b). β-actin was used as the loading control. Effect of low or high concentrations of AECS/p-BQ on apoptosis in cultured A549 cells and its prevention by vitamin C as evidenced by flow cytometry (c). A549 cells were grown on 60 mm culture plates and were gradually serum starved for 3 days to synchronize the cells. Then the cells were either nontreated (NT) or exposed to 2 μL/mL or 50 μL/mL AECS for 1 hr, 200 ng/mL or 2.5 μg/mL p-BQ for 1 hr, with or without 40 μg/mL vit C pretreatment in serum-free medium for 15 min. After treatment, cells were incubated in fresh media containing serum for 12 hr, followed by Annexin V-PI assay. Bar graphs show the percentage of normal and apoptotic cells after respective treatments, as evidenced by flow cytometry (d). The numbers within the bars represent percentage of normal and apoptotic cells.