Figure 1.

Nickel induces apoptosis. (A) BEAS-2B cells (4 × 10⁵) were seeded in a 60 mm dish overnight, then treated without or with various concentrations of Ni₃S₂ for 48 h, and then stained with Annexin V/PI. Apoptosis was determined using flow cytometry as described in the Materials and Methods. Illustrated is a representative of at least three separate experiments. (B) Quantification of apoptotic cells. Data were obtained from Annexin V/PI assays and are represented as means ± SEs of three separate experiments. *P < 0.05 as determined by the treatment vs control. (C) BEAS-2B cells (1.5 × 10⁵) were seeded in each well of six well plates overnight and then treated without or with various doses of Ni₃S₂ for 72 h or with 2 μg/cm² Ni₃S₂ for various time points as indicated, and then, cell counting was carried out. Data represent means ± SEs of three individual experiments of cell counting. *P < 0.05 as determined by the treatment vs control. (D) Nickel induces morphological changes in BEAS-2B cells. BEAS-2B cells were seeded in 60 mm dishes. After they were cultured at 37 °C overnight, the cells were treated without or with 2 μg/cm² Ni₃S₂ for 48 h. Pictures were taken using a phase contrast microscope. (E) Down-regulation of Bcl-2 and Bcl-xL is involved in nickel-induced apoptosis. BEAS-2B cells were seeded in each 100 mm dish and cultured in 10% FBS/DMEM at 37 °C. When the cell density reached 70−80%, the cells were exposed to different concentrations of Ni₃S₂ for 48 h. After treatments, total cellular extracts were prepared and subjected to Western blot assay using antibodies against Bcl-2, Bcl-xL, and β-actin. Each lane was loaded with 40 μg of protein. Blots were subsequently stripped and reprobed with antibody against β-actin to ensure equivalent loading and transfer. The results shown are representative of three separate experiments. Two additional studies yielded equivalent results. (F) Band densities in the Western blots were normalized against β-actin. Each bar represents the mean ± SE of the three independent experiments. All means marked with * (P < 0.05) are significantly different from the control.