CE-induced apoptosis is caspase-3 dependent.

<p>(A) CE instigates apoptosis in BEAS-2B cells. Cells were pretreated with or without 10 μM ZnPP for overnight. Following exposure of BEAS-2B cells to 0 ppm, 150 ppm or 300 ppm (data not shown) of CE for 1 or 4 hours, flow cytometry dot plots for the simultaneous binding of Annexin V-FITC and PI uptake were shown. Numbers in the gates represent percentages of Dead (D) cells, as well as early (E), and late (L) apoptotic events. The data are representative of three independent experiments. Percentages of dead cells (PI⁺Annexin V^-) and apoptotic cells (Annexin V⁺) were quantified and data are shown as a mean ± SD. * <i>p</i> < 0.05, ** <i>p</i> < 0.01 vs no CE control in the absence of ZnPP; # <i>p</i> < 0.05, ## <i>p</i> < 0.01 vs no CE treatment in the presence of ZnPP; & <i>p</i> < 0.05, && <i>p</i> < 0.01 vs with CE treatment in the absence of ZnPP by a one-way ANOVA with HSD test. (B) Representative western blots and associated quantification for active caspase-3, normalized by β-actin content. BEAS-2B cells were exposed to 0 to 150 ppm of CE for 4 hours. Antibodies specific cleaved caspase-3 was used and β-actin was used as a loading control. The representative blots from three independent experiments are shown. The densities of protein bands were determined by densitometry and the data represent a one-fold increase from the control density. (C) Caspase-3 activity was measured using a DEVD-pNA calorimetric assay. After treatment with different concentration of CE for 4 hours, cells were lysed and 100 μg of protein was incubated with 200 μM DEVD-pNA for 6 hours at 37°C. Absorbance measurements were taken at a wavelength of 405 nm and the fold induction of caspase-3 activity relative to the control was shown. * <i>p</i> < 0.05 and ** <i>p</i> < 0.01 vs control by a one-way ANOVA with HSD test. (D) Mice tracheal explants were isolated and IHC analysis was performed after exposure 0 ppm or 150 ppm CE for 2 hours. (E) Blue crabs were exposed to 0 ppm or 150 ppm CE for 19 hours. Gill tissues were harvested for IHC analysis using a cleaved caspase-3 polyclonal antibody (1:800 dilution) followed by treatment with biotinylated goat anti-rabbit antibody and streptavidin–alkaline phosphatase (AP), which produced a red coloration for cleaved caspase-3 positive areas.