Cleaved caspase 3 c casp 3

Cisplatin (DDP, Cat.^#H20010743) injection was purchased from Jiangsu Hausen Pharmaceutical Co., Ltd (Jiangsu, China). Dulbecco's modified Eagle's medium (DMEM, Cat.^#11965092), fetal bovine serum (FBS, Cat.^#10270106), penicillin/streptomycin (Cat.^#10378016), and phosphate-buffered saline (PBS, Cat.^#10010023) were supplied by Gibco (NY, USA). Primary antibodies against Beclin1 (Cat.^#11306-1-AP), p62 (Cat.^#66184-1-Ig), Cytochrome c (cyto. C, Cat.^#66264-1-Ig), and DAPK1 (Cat.^#25136-1-AP) were purchased from Proteintech (Wuhan, China). Primary antibodies against Phospho-AMPKα (p-AMPKα, Cat.^#50081S), Phospho-mTOR (p-mTOR, Cat.^#5536T), Atg7 (Cat.^#8558T), LC3A/B (Cat.^#12741S), Cleaved Caspase-9 (c-Casp 9, Cat.^#20750S), Cleaved Caspase-3 (c-Casp 3, Cat.^#9661T), Cleaved PARP (c-PARP, Cat.^#5625T), Phospho-SAPK/JNK (p-JNK, Cat.^#9255S), Phospho-p53 (p-p53, Cat.^#9284T), Bax (Cat.^#2772T), and GAPDH (Cat.^#5174T) and rabbit (Cat.^#7074P2) or mouse (Cat.^#7076P2) secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Cells were fixed with 4 % paraformaldehyde for 10 min at room temperature (RT). Staining was done with antibodies against CD68 (BD Biosciences, Heidelberg, Germany), cleaved caspase 3 (cCasp-3) (Cell Signaling Technology, Leiden, Netherlands), and goat-anti-rabbit-Cy3 (Dianova, Hamburg, Germany) and goat-anti-mouse-AlexaFluor488 (AF488) as secondary antibodies, and DAPI (Life Technologies, Karlruhe, Germany). Samples were embedded into fluorescent mounting medium (Dako, Hamburg, Germany). Subsequently, imaging was performed on an AXIO Observer Z1 fluorescence microscope equipped with Apotome 2 (Carl Zeiss AG, Jena, Germany).