Evidence-Based Complementary and Alternative Medicine / 2011 / Article / Fig 4

Original Article

Agaricus blazei Extract Induces Apoptosis through ROS-Dependent JNK Activation Involving the Mitochondrial Pathway and Suppression of Constitutive NF-κB in THP-1 Cells

Figure 4

The role of ROS on ABE-mediated apoptosis. THP-1 cells (2 × 105 cells ml−1) were pretreated with 10 mM NAC, and then 4 mg ml−1 of ABE was treated for 12 h (a) and 24 h (b–d). (a) The redox status was monitored by the oxidation-sensitive fluorescent dyes for and H2O2. The mitochondrial membrane potential was also determined by flow cytometer using DiOC6 labeling of mitochondria. (b) DNA contents were analyzed by flow cytometer. The sub-G0/G1 DNA fractions, indicating apoptosis, were determined as a percentage of the total number of cells. (c) Equal amounts of cell lysate were resolved on SDS-PAGE, transferred to nitrocellulose membranes, and probed with specific antibodies for cytochrome c, Bax, and PARP. β-Actin was used as an internal loading control. (d) THP-1 cells were collected following 24 h exposure to 4 mg ml−1 of ABE with or without 2 h of pretreatment with NAC. Caspase-3 activity was determined following the manufacturer's protocol. Each point represents the mean ± SD of three independent experiments. The significance was determined by Student's t-test ( versus vehicle control). Cyto, Cytoplasm.
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