Evidence-Based Complementary and Alternative Medicine / 2013 / Article / Fig 3

Research Article

Antcin C from Antrodia cinnamomea Protects Liver Cells Against Free Radical-Induced Oxidative Stress and Apoptosis In Vitro and In Vivo through Nrf2-Dependent Mechanism

Figure 3

Antcin C promotes transcriptional activation of Nrf2 in AAPH-induced HepG2 cells. (a) Cells were pretreated with antcin C (20 μM) or silymarin (100 μM) for 2 h, and then exposed to AAPH (10 mM) for 1 h. Nuclear and cytoplasmic lysates were prepared and subjected to western blot analysis. The accumulation of Nrf2 in the cytoplasm and the nucleus was monitored. (b) HepG2 cells were pretreated with antcin C (5–20 μM) or silymarin (100 μM) for 2 h, and then exposed to AAPH (10 mM) for 1 h. The protein expression levels of Nrf2 in AAPH-treated HepG2 cells were measured by immunofluorescence using Nrf2 specific primary and fluorescein isothiocyanate-conjugated secondary antibodies (green). The subcellular and nuclear distribution was photographed by fluorescence microscope. DAPI (1 μg/mL) was used to stain the nucleus. (c) HepG2 cells were transiently transfected with ARE plasmids by using lipofectamine and pre-incubated with antcin C (20 μM) or silymarin (100 μM) in the presence or absence of AAPH (10 mM) for 2 h. Cell lysates were mixed with luciferase reagents and quantified using an illuminometer. Relative ARE activity was calculated by dividing the relative luciferase unit (RLU) of treated cells by the RLU of untreated cells. Values represent the mean ± SD of three independent experiments. was considered significant for sample versus AAPH. was considered significant for control versus AAPH. (d) HepG2 cells were transfected with a specific siRNA against Nrf2 or a nonsilencing control. After 24 h of transfection, the cells were incubated with or without antcin C (20 μM for 2 h) and were induced by AAPH (10 mM) for 2–24 h. Protein expression levels of HO-1, NQO-1, and Nrf2 were monitored by western blot analysis. Cell viability was determined by MTT assay. Values represent the mean ± SD of three independent experiments. was considered significant for AAPH versus sample in the control siRNA. was considered significant for AAPH versus sample in the siNrf2 cells.
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