During the experiment, body weight was measured under the same environment. It was measured every Monday and Thursday at 9:00 AM (twice a week).

To determine protein levels, liver tissue was homogenized in 0.5 M EDTA (Duksan, Korea), lysis buffer (Gendepot, R4200-100, USA), and phosphatase inhibitor 100 x (Gendepot, P3200-001, USA). After homogenization, each sample was centrifuged at 1,200 x g for 10 minutes. Supernatants were collected and subjected to protein concentration measurement using Bradford protein assay method. Briefly, standard BSA or supernatant (1 ㎕) was mixed with 1 ml of Bradford reagent. A 200 ㎕ of the mix was then placed in an ELISA plate to measure the absorbance value at wavelength of 595 nm. After protein concentration measurement, proteins were mixed with Laemmli sample buffer and boiled for 5 min. Protein samples (20μl each) were then loaded into 10% SDS-PAGE gel and run at 100 V to separate the proteins. After electrophoresis, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes by electro-blotting at 100 V at room temperature for 1 h (± 20~30 min). The membranes were incubated with 5% skim milk blocking buffer (Skim milk block buffer; TBST+5% skim milk) for 1 h followed by incubation with primary antibodies at 4℃ overnight. Primary antibodies were prepared by diluting in 5% skim milk. The following primary antibodies were used: HSP70 1:2,000 (Abcam, UK), Bcl-2 1:200 (Santa cruz, USA), p53 1: 1,000 (Abcam, UK), and caspase-3 1:1,000 (Cell signaling, USA). After washing 5 times (10 min each) with TBST buffer (50 mM Tris-Hcl, 150 mM NaCl and 0.05%, Tween 20), membranes were incubated with respective secondary antibodies at room temperature for 1 h. For HSP70 1:5,000, goat-anti rabbit secondary antibody (Santa Cruz, USA) was used. For Bcl-2 1:2,000, goat-anti rabbit secondary antibody (Santa Cruz, USA) was used. For p53 1:5,000, goat-anti mouse secondary antibody (Santa Cruz, USA) was used. For caspase-3 1:3,000, goat-anti rabbit secondary antibody (Santa Cruz, USA). After rinsing with TBST (5% tween-20) 5 times (10 min each), the membranes were incubated with ECL reagent and developed onto X-ray film. Band intensities were measured with Image J (NIH, Ver. 1.47t, USA). Their intensities were normalized to the level of β-actin as loading control.

Blood cortisol concentrations were measured using an ELISA kit (MyBioSource, CANADA). Briefly, blood samples were centrifuged at 1,000 rpm for 10 minutes and left at room temperature for 20 minutes after diluting the sample with reagent provided by the kit in accordance with the manufacturer’s instructions. Both standard and sample dilutions were performed in 96-well plates. They were incubated at 37℃ after mixing 100 ㎕ for 90 minutes in a shaker. Washing was then performed with buffer three times, followed by the addition of 400 ㎕ to each well after mixing. The plate was turned upside down to remove the wash buffer. Then 100 ㎕ of prepared antibody was added to each well and incubated at 37℃ with shaking for 60 minutes. The plate was then washed with washing buffer three times. Cortisol enzyme-conjugate mixed with buffer (100 ㎕) was then added to each well followed by incubation at 37℃ for 30 minutes with shaking. Finally, washing was performed using 400 ㎕ of washing buffer five times. The plate was turned upside down to remove the wash buffer. After adding 100 ㎕ of a dark color reagent, the 96-well plate was placed on a shaker for mixing, followed by addition of 100 ㎕ of color reagent C. After 30 minutes of mixing, the absorbance value was measured at wavelength of 450 nm on an ELISA reader (Tecan Infinite, F50, Austria).