Pcdna3.1 ha vector

The human ESCC cell line (EC9706) was obtained from the American Type Culture Collection (ATCC, Rockville, Maryland, USA) and kept in RMPI 1640 medium with 10% fetal bovine serum (FBS) (Gibco, California, USA), 1% of 100 U/mL penicillin, and 1% of 100 mg/mL streptomycin sulfate. The cells were incubated in humidified incubators with 5% CO² at 37°C. The cells were first treated with different concentrations of propofol to test the effect of propofol on EC9706 cells. Then, EC9706 cells were transfected with SOX4-siRNA or DNA3.1+HA-SOX4 to decrease or increase the expression of SOX4 before propofol was added for testing whether SOX4 plays the key role in propofol’s effect on EC9706 cells.
NC-siRNA and SOX4-siRNA were synthesized chemically at Suzhou GenePharma Co. Ltd. (Suzhou, China). Human SOX4 gene was constructed into pcDNA3.1+HA vector by Life Technologies (Invitrogen, California, USA), and the empty vector served as the negative control. For transfection, after the cells were cultured to 70–80% confluence, NC-siRNA or SOX4-siRNA was transfected by using Lipofectamine 2000 (Invitrogen, California, USA) according to the manufacturer’s instructions for inhibition of the expression of SOX4 in the cells. Meanwhile, pcDNA3.1+HA-SOX4 or pcDNA3.1+HA empty vector was transfected by using Lipofectamine 2000 for over-expression of SOX4 in the cells.