Cell culture and reagents

All ESCC cell lines (KYSE30, KYSE70, KYSE140, KYSE150, KYSE410, KYSE450, KYSE510 and EC109) and 293T cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and were cultured at 37°C in a 5% CO₂ humidified incubator and were not maintained for >2 months. All ESCC cells were maintained in RPMI-1640, while 293T cells were cultured in DMEM supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml) and 10% FBS (Biological Industries). All cell lines were mycoplasma-free and were authenticated using short tandem repeat DNA typing. The human immortalized normal esophageal epithelial cell line SHEE was kindly gifted by Dr Enmin Li from the Laboratory of Tumor Pathology (Shantou University Medical College, Shantou, Guangdong, China). Transfection was performed using Lipofectamine^® 2000 (cat. no. 11668-019; Invitrogen; Thermo Fisher Scientific, Inc.) for ESCC cell lines and Simple-Fect Reagent (Signaling Dawn Biotech) for 293T cells according to the manufacturer's protocol. AURKA inhibitor alisertib was purchased from Selleck Chemicals (cat. no. S1133).

The antibodies used in the present study were as follows: Anti-AURKA (cat. no. ab13824; Abcam), anti-Flag (cat. no. F1804; MilliporeSigma), anti-HA (cat. no. 3724; Cell Signaling Technology, Inc.), anti-His (cat. no. ab137839; Abcam), anti-phosphorylated (p)-PI3K (Tyr467) (cat. no. sc-293115; Santa Cruz Biotechnology, Inc.), anti-p-AKT (Ser473) (cat. no. 4060; Cell Signaling Technology, Inc.), anti-p-Akt (Thr308) (cat. no. 13038; Cell Signaling Technology, Inc.), anti-PI3Kp85 (cat. no. 4257; Cell Signaling Technology, Inc.), anti-Akt (pan) (cat. no. 4691; Cell Signaling Technology, Inc.), anti-p21 (cat. no. sc-397; Santa Cruz Biotechnology, Inc.), anti-p27 (cat. no. 3686; Cell Signaling Technology, Inc.), anti-p53 (cat. no. sc-126; Santa Cruz Biotechnology, Inc.), anti-TPX2 (cat. no.122455; Cell Signaling Technology, Inc.), anti-RPS6 (cat. no. 2217; Cell Signaling Technology, Inc.), anti-HSP27 (cat. no. sc-13132; Santa Cruz Biotechnology, Inc.), anti-VDAC (cat. no. 4866; Cell Signaling Technology, Inc.), anti-RPL7 (cat. no. 2415; Cell Signaling Technology, Inc.) and anti-GAPDH (cat. no. HRP-60004; ProteinTech Group, Inc.).