Western blot analysis (WB)

To extract total protein, RIPA Lysis Buffer (P0013B, Beyotime Biotechnology, China) with protease inhibitor phenylmethanesulfonyl fluoride (PMSF; ST506, Beyotime Biotechnology, China) and protease inhibitor cocktail (K1011, APExBIO Technology LLC) were added into tissues and cells. The homogenate was centrifuged at 14,000×g for 45 min at 4 °C. 40 μg total protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane. 5% bovine serum albumin (BSA) was used to block the membranes for 2 h at room temperature. Then, the membranes were incubated with specific primary antibodies overnight at 4 °C. Then, the membranes were incubated with secondary antibodies for 2 h at room temperature. Antibodies used in this study included the following: rabbit polyclonal anti-DTX3 (1:1000, cat. no. 25304-1-AP, Proteintech Group Inc), rabbit polyclonal anti-Vimentin (1:1000, cat. no. 10366-1-AP, Proteintech Group Inc), mouse monoclonal anti-E-cadherin (1:2000, cat. no. 60335-1-Ig, Proteintech Group Inc), rabbit polyclonal anti-N-cadherin (1:2000, cat. no. 22018-1-AP, Proteintech Group Inc), rabbit monoclonal anti-AKT (1:1000, cat. no. #4691, Cell Signaling Technology Inc), rabbit monoclonal anti-Phospho-AKT (1:2000, cat. no. #4060, Cell Signaling Technology Inc), rabbit monoclonal anti-NDUFAF5 (1:1000, cat. no. ab192235, Abcam Inc), rabbit polyclonal anti-GAPDH (1:10000, cat. no. 10494-1-AP, Proteintech Group Inc), rabbit monoclonal anti-Histone H3 (1:2000, cat. no. #4499, Cell Signaling Technology Inc), rabbit polyclonal anti-tubulin (1:10000, cat. no. 11224-1-AP, Proteintech Group Inc), rabbit polyclonal anti-XRCC5 (1:1000, cat. no. 16389-1-AP, Proteintech Group Inc), rabbit polyclonal anti-ubiquitin (1:1000, cat. no. 10201-2-AP, Proteintech Group Inc) and peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (H+L) (1:3000, Zhongshan Goldenbridge Company, Beijing, China) as the secondary antibody. Enhanced chemiluminescence kit (P0018AS, Beyotime Biotechnology, China) was performed to show the binding conditions between antigens and antibodies. The integrated optical density (IOD) of each band was measured with the Image-Pro Plus 6.0 software (Media Cybernetics Inc., Rockville, MD). The ratio between the IOD of target protein and internal control in the same sample was calculated as the relative protein expression of the target protein.