Cell apoptosis analysis kit

For the cell viability assay, 10,000 cells per well were seeded into a 96-well plate and cultured overnight. Following treatment with 3-MA or LY294002, 10 μl CCK-8 solution was added to each well and the optical density 450 absorbance was measured by a multifunctional microplate reader (Thermo Fisher Scientific) after a 2-h incubation at 37°C. For the cell apoptosis assay, the cells were stained with Annexin V/propidium iodide (PI), and cell apoptosis was analyzed using flow cytometry. In accordance with the instruction of cell apoptosis analysis kit (EnoGene), cells (5×10⁵) were harvested and centrifuged at 1,000 × g for 5 min. Cell samples were then resuspended in 500 μl of binding buffer. Annexin V-enhanced green fluorescent protein (5 μl) and PI (5 μl) were added to the samples and incubated in the dark for 15 min. A total of 1×10⁴ cells were collected per sample and detected on FACSCalibur (BD Biosciences, San Jose, CA, USA). The data were finally analyzed using FlowJo software (FlowJo LLC, Ashland, OR, USA).

RPMI-1640 medium was purchased from Gibco; Thermo Fisher Scientific (Grand Island, NY, USA). The autophagy inhibitors 3-methyladenine (3-MA) and LY294002 were obtained from Sigma-Aldrich; Merck KGaA (St. Louis, MO, USA). Rabbit anti-human/mouse/rat polyclonal antibody LC3 (E18-5402, 1:500), GAPDH (E1C604, 1:2,000), cytochrome oxidase (COX) IV (E12-327, 1:1,000), cleaved caspase-3 (E11-0104L, 1:500), cleaved caspase-8 (E18-5267, 1:1,000), cleaved caspase-9 (E18-5240, 1:1,000), mouse anti-human/mouse monoclonal antibody Bcl-2 (E10-30077, 1:1,000), rabbit anti-human/mouse polyclonal antibody Bax (E11-0773B, 1:500), Beclin-1 (E90562, 1:500), mouse anti-human/mouse/rat monoclonal antibody cytochrome c (E12-378, 1:2000), goat anti-rabbit (E0L3012), goat anti-mouse IgG secondary antibodies (E0L3032), Cell Counting Kit-8 (CCK-8) and cell apoptosis analysis kit were all purchased from EnoGene (New York, NY, USA). Mouse anti-human p53 polyclonal antibody was obtained from BD Pharmingen (554294; San Jose, CA, USA). TRIzol reagent was obtained from Life Technologies; Thermo Fisher Scientific (Carlsbad, CA, USA). RevertAid First Strand cDNA Synthesis kit and QuantiFast SYBR Green PCR kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Mitochondrial membrane potential assay kit with JC-1 was obtained from Beyotime Biotechology (Shanghai, China).