Cell counting kit 8 cck 8

Cell viability assay was performed with a Cell Counting Kit-8 (CCK-8; EnoGene, Nanjing, China) according to the manufacturer's instructions. Briefly, cells were plated into 96-well plate at a concentration of 5 × 10³ cells per well. After incubation for indicating time periods, each well was added in 10 μl CCK-8 and 90 μl DMEM. The absorbance was measured at 450 nm after incubation for 40 min at 37 °C.
Migration assay were performed as described previously [26 (link)]. Briefly, 1 × 10⁵ of HCCLM-3 cells in FBS-free medium were seeded in the upper transwell chamber (Corning, New York, USA), and 600 μL DMEM with 20% FBS was added in the lower chamber. After incubation for 24 h at 37 °C, cells on the bottom surface of the chamber were fixed using 4% paraformaldehyde (Biosharp, Beijing, China) for 15 min and stained with 0.1% crystal violet for 20 min, while the top surface of the chamber was wiped by a cotton swab to remove cells. The number of migrated cells were imaged and counted.

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).