β action

Ovarian tissue samples were homogenized in PBS with a Teflon-glass homogenizer (Thomas Scientific, Swedesboro, NJ, USA) on ice and sonicated. Protein concentrations were determined by BCA protein assay (TIANGEN BIOTECH, Beijing, China). The protein samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes (BioTrace™ NT, Port Washington, NY, USA). The membranes were blocked in 5% nonfat dry milk in tris-buffered-saline with tween 20 (TBST) for 1 h and incubated with a primary antibody against SIRT1, SIRT6, and FOXO3a (1:200 dilution, Santa Cruz Biotechnology, CA, USA), NRF1 (1:400 dilution, Santa Cruz Biotechnology, CA, USA), p53 (1:600 dilution, Santa Cruz Biotechnology, CA, USA) or β-action (1:1,000 dilution, Santa Cruz Biotechnology, CA, USA) over-night at 4°C, followed by the incubation with a secondary (horseradish peroxidase-conjugated) anti-rabbit or anti-mouse antibody (1:5,000 diluted) at room temperature for 1 h. Bands were visualized with a chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA, USA). Band intensities were analyzed using the Quantity One software (Bio-Rad Laboratories Pty. Ltd., Hercules, CA, USA). β-actin was used as a loading control.

Cells were washed twice with ice-cold PBS and solubilized in RIPA buffer (Sigma-Aldrich, Saint Louis, MO) on ice and then was quantified using QuantiPro bicinchoninic acid assay kit (Sigma-Aldrich). Proteins were denatured at 100°C with sample buffer for 5 min. Equal amounts of protein (50 μg) separated by electrophoresis in 10% to 12% SDS-PAGE gels according to their molecular weight. Proteins were transferred onto PVDF membranes (Bio-Rad) and blocked for 2 h in blocking solution (5% nonfat dry milk in TBS containing 0.1% Tween 20). The membrane was then exposed to the primary antibody overnight at 4°C. Snail, slug, NF-κB, p-NFκB P65 (Ser536), STAT3, p-STAT3, AKT, p-AKT were purchased from Cell Signaling (Beverly, MA); Twist1, Zeb1, Zeb2, vimentin, E-cadherin, N-cadherin, β-action were purchased from Santa-Cruz Biotechnology, all primary antibodies dilution is 1:1000. After washing, the membranes were incubated for 1.5 h at room temperature with peroxidase-linked secondary antibody (Santa-Cruz). Signals were revealed with an electrochemoluminescence (ECL) Western Blotting Analysis System (Pierce) using the FluorChem®FC2 (Alpha Innotech, CA), and then quantified using ImageJ® software.