Apoptotic and Autophagic Effects of Sesbania grandiflora Flowers in Human Leukemic Cells
Figure 4
Involvement of mitochondrial pathway in F2 induced apoptosis.
(A) Loss of mitochondrial membrane potential. U937 cells (2.5×105/ml) were incubated with F2 (18.6 µg/ml for 6 h, 12 h, 24 h, 48 h). Cells were loaded with mitochondrial sensor dye JC-1 (7.5 µM; 15 min) as described in Materials and methods and analysis was revealed by the shift from red to green fluorescence in time dependent manner. Data is a representative of three different experiments. (B) The expression levels of pro- and anti-apoptotic proteins. Whole cell extracts were made from control and F2 treated U937 cells (2.5×105/ml). Equal amounts of cell lysates were resolved by SDS-PAGE, transferred to PVDF membrane and probed with specific antibodies against Bcl-2, Bax. Analysis was confirmed with three different sets of experiments. (C) Effect of F2 on release of cytochrome c into cytosol. Cytoplasmic and mitochondrial fractions were prepared from control and F2 treated (18.6 µg/ml for 6 h, 12 h, 24 h, 48 h) U937 cells using mitochondria/cytosol fractionation kit and cytochrome c was analyzed by Western blotting with anti cytochrome c antibody. Data shown are from one of the three experiments. β-actin served as a loading control.