Cytochrome c

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).

Cells are treated with chloroquine for 12-24 hours, the cell pellet is collected by centrifugation, add lysis buffer (Beyotime Biotechnology, Shanghai, China), and protein is collected. After the animal experiment treatment is over, the subcutaneous tumor is taken out, the tissue is frozen and ground with liquid nitrogen, the lysate is added, and the tissue protein is extracted. Use the BCA protein quantification kit (Beyotime Biotechnology, Shanghai, China) to determine the protein concentration and determine the sample volume. After the electroporation is over, 5% skim milk is blocked at room temperature for 1-2 hours, and the primary antibody is incubated at 4°C overnight. The primary antibodies are as follows: PD-1(1:1000, Abcam, MA), MMP2,Cleaved-caspase-3,PD-L1,CD4,CD8 (1:1000,CST,USA), Bax, Bcl-2, Cytochrome-C(1:1000, Abways, China), and Tubulin (1:3000,Sigma,USA), After incubating for 1 hour with the secondary antibody corresponding to the primary antibody, wash the PVDF membrane (Millpore, USA) and use ECL chemiluminescent solution (Millpore, USA) for chemical imaging. Use Image J software for gray value analysis.