Methanolic Extract of Artemia salina Eggs and Various Fractions in Different Solvents Contain Potent Compounds That Decrease Cell Viability of Colon and Skin Cancer Cell Lines and Show Antibacterial Activity against Pseudomonas aeruginosa
Figure 6
Hexane and chloroform fractions of the methanolic extract induced apoptosis in HCT116 and B16-F10 cells. (a) Intracellular ROS generation was determined by treating cells with 100 µg/ml hexane and chloroform fractions for 24 h and then incubating them with DHR123 for 20-30 min. Images were obtained using a fluorescence microscope, with excitation and emission wavelengths of 500 and 536 nm, respectively. 5-FU was used as standard. (b) Cells were treated with 10 μg/ml hexane and chloroform fractions. Cells were collected, fixed with 70% ethanol overnight at −20°C, and then stained with PI. DNA content was analyzed by flow cytometry. 5-FU was used as standard. (c) Cellular proteins were extracted using cell lysis buffer. Proteins were quantified by Bradford assay, and equal amount of proteins was loaded onto SDS-PAGE gels. Electrophoresis was conducted at a constant voltage of 100V for 140 min. Proteins were then transferred onto nitrocellulose (NC) membranes. NC membranes were blocked with 3% BSA for 1 h at room temperature. NC membranes were then incubated with specific antibodies (1:1000) for cleaved caspase-3 and cleaved PARP overnight at 4°C. NC membranes were washed with TBST for 40 min and then incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (1:2000). Chemiluminescent signals were developed using Clarity™ ECL Western Blotting Substrate. Actin was used as internal control in all experiments.