KYSE30 and KYSE410 cells in the logarithmic growth phase were adjusted to a cell density of 3×104 cells/ml, and seeded into 96-well cell culture plates, with 100 µl per well and three replicates per group. Until the cells were attached, 20 µl serum-free RPMI-1640 medium was used to dilute shRNA (0.15 µg) and Lipofectamine® 2000 (0.45 µl) for transient transfection. After 48 h of transfection of ESCC cells, 10 µl CCK-8 reagent was added to each well (110 µl/well) of a 96-well plate and incubated for 3 h in a 5% CO2 incubator. Then, the absorbance of 450 nm water soluble tetrazolium salt was measured at 0, 24, 48 and 72 h to quantify cell proliferation.

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