Cleaved parp

Total protein was extracted from cells using RIPA buffer (Beyotime) at 4 °C about 20 min while the PMSF (Beyotime) was used for the inhibition of protein degradation. Equal amounts of sample protein (15–20 µg per lane) were separated by SDS-PAGE and transferred onto 0.45 µm PVDF membranes (Millipore). After blocked with 5% milk at 20 °C about 1 h, membranes were incubated with the corresponding primary antibody at 4 °C overnight, followed by three times wash in TBST for 10 min. After that, the membranes were incubated with HRP-conjugated secondary antibodies (Beyotime) at 20 °C. Protein bands were exposed by the Gel Doc 2000 (BioRad). The primary antibodies mentioned above were for: SLC25A22 (Abcam, 1:1000), E-cadherin (Abways, 1:1000), N-cadherin (Abways, 1:1000), vimentin (CST, 1:1000), ERK (CST, 1:1000), p-ERK (CST, 1:1000), MEK (CST, 1:1000), p-MEK (CST, 1:1000), GAPDH (Abways, 1:1000), BCL-2 (ABclonal, 1:1000), cytochrome-c (CST, 1:1000), PARP (CST, 1:1000), cleaved PARP (CST, 1:1000).

The CNE-2 and CNE-2R cells, after treatment, were extracted with the buffer radioimmuno- precipitation assay (RIPA), supplemented with protease and phosphatase inhibitors (Roche, NJ, USA). To determine the protein concentrations, we used the Bradford Protein Assay Kit. The proteins were then separated using SDS-PAGE at 12% or 15% and transferred to PVDF membranes. After blocked, these membranes were incubated overnight at 4°C with the corresponding primary antibodies. Cell Signaling Technology provided anti-LC3 (#4108), P62 (#88588), caspase-3 (#9662), cleaved caspase-3 (#9664), Survivin (#2808), JNK (#9252) and phospho-JNK (#4668). Cleaved PARP (#P09874) was purchased from Abways Technology. Bcl2 (sc-130308) and Bax (sc-6236) were purchased from Santa Cruz Biotechnology. GADPH and β-actin primary antibodies were obtained from Proteintech, China. The bands of protein were developed with a chemiluminescence reagent (Cell Signaling).