Ecl assay

Protein extracts were resolved by 8%–15% SDS-PAGE, transferred to polyvinylidene fluoride membranes, and probed with antibodies against human VE-cadherin (CST), vimentin (CST), fibronectin (Abcam), Snail (CST), PITPNM3 (Santa Cruz, USA), and GAPDH (Santa Cruz), as well as against ERK, AKT, GSK-3β, and the corresponding phosphorylated proteins (CST). Peroxidase-conjugated anti-mouse or anti-rabbit IgGs (CST) were used as secondary antibodies, and the antigen-antibody reactions were visualized by enhanced chemiluminescence (ECL) assay (Thermo, USA).

Total cell lysates were prepared with 1% SDS and 10 mM Tris buffer (pH 7.4), and separated by SDS-PAGE. After transfer to a nitrocellulose membrane, the proteins were immunoblotted with different antibodies overnight at 4°C. The following antibodies were used: anti-ubiquitin (catalog # 3933) (1:1000), anti-p21 (catalog #: 2947) (1:500), anti-survivin (catalog #: 2808) (1:2000), anti-cleaved PARP (catalog #: 9546) (1:300), and anti-cyclin B1 (catalog #: 12231) (1:1000), all from Cell Signaling Technology (Boston, MA); anti-aurora A from Abcam (Cambridge, MA) (catalog #: ab190367) ; and anti-β-actin (catalog #: sc-81178) (1:3000) from Santa Cruz Biotechnology (Dallas, TX). Anti-human β-actin was used as a loading control. The membranes were incubated with the appropriate HRP-conjugated IgG (anti-rabbit antibody at 1:3000 dilution, Cell Signaling Technology, Danvers, MA, USA, or anti-mouse antibody at 1:10 000 dilution, Santa Cruz Biotechnology). An ECL assay (Thermo Scientific, Rockford, IL, USA) was used to detect the proteins.

Liver tissue samples and cultured cells were lysed in RIPA buffer. The nuclear and cytoplasmic protein fractions were extracted using a nuclear and cytoplasmic protein extraction kit (Beyotime, Shanghai, China) according to the manufacturer’s protocol. Protein concentration was measured using a BCA protein assay kit (Boster, Wuhan, China). The protein samples were separated by SDS–PAGE (Bio–Rad Laboratories Inc., Berkeley, CA, USA) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore Corp, Billerica, MA, USA). The PVDF membranes were incubated with primary antibodies against PGC-1α, BNIP3, Beclin-1, MCAD, CPT-1α (1:500; Proteintech, Wuhan, China), PPAR-α (1:500; Cell Signaling Technology, USA), Hif-2α and LC3B (1:1000; Abcam, Cambridge, MA, USA), and Lamin A/C (1:500; Zhongshan Jinqiao, China) for 24 h, and then incubated with secondary antibody (1:500; Zhongshan Jinqiao, China) for 1 h at room temperature. The protein bands were visualized by enhanced chemiluminescence (ECL) assay (Thermo Scientific, USA), and the blots were analyzed using Image Lab 3.0 (Bio–Rad, Hercules, CA, USA). The gray value of protein bands was calculated using Image J 1.50i (National Institutes of Health, USA) and normalized to that of the internal control, β-actin.