*2.6. Apoptosis*

The cell apoptosis was determined using a commercial Annexin V-FITC apoptosis detection kit (Invitrogen, Carlsbad, CA, USA). After the treatment of different chemicals, the hepatocytes were incubated with 100 μL 1× binding buffer containing 5 μL Annexin-V-FITC and 1 μL propidium iodide (PI) for 30 min at room temperature. After the incubation, 400 μL binding buffer was added to the culture to stop the staining. The flow cytometric analysis was then performed. Data was analyzed using Flowjo 7.6 software.

#### *2.7. Mitochondrial Permeability Transition Pore (mPTP) Opening*

The mPTP opening was examined using the commercial kit by monitoring the release of calcein from mitochondrial. Briefly, the hepatocytes were treated with 2 μM calcein-AM and 1 mM CoCl2 for 30 min at room temperature, and washed with PBS. The culture was then incubated with 1 mM CoCl2 for an additional 20 min at 37 ◦C in order to specifically quench the fluorescence of free calcein in the cytosol. The fluorescence intensity of mitochondrial calcein in L02 hepatocytes was determined using a fluorescence microplate reader at 490 nm/515 nm for excitation/emission. The loss of calcein fluorescence suggested the opening of mPTP.

#### *2.8. Mitochondrial Membrane Potential (MMP,* Δψ*m)*

The MMP of L02 hepatocytes was detected using JC-1 (Sigma, St. Louis, MO, USA). After the chemicals treatment, the cells were washed with PBS and then incubated with JC-1 for 20 min at 37 ◦C. The fluorescence intensity was read at the excitation/emission wavelength of 488/530 nm. The MMP is presented as % of control for the fluorescence intensity.
