HMLER cells were seeded at 2000 cells per well in 96-well plates. Twenty-four hours later, the cells were treated for 48 hours with doxorubicin, cisplatin, cyclophosphamide, 5-fluoruracil, or paclitaxel at the indicated concentrations. Cell viability was measured using Cell Count Reagent SF (Nacalai Tesque) according to the manufacturer’s instruction. The reagent was added to the cells, and the cells were incubated at 37°C for 4 hours. Absorbance at 450 nm was measured with a Model 680 microplate reader (Bio-Rad), followed by subtraction of absorbance at 595 nm. For the cell survival and proliferation assay, HMLER cells were seeded at 10,000 cells per well in 24-well plates. Twenty-four hours later, the cells were incubated with anticancer drugs, TGF-β, or AKT-mTOR inhibitors for 48 hours. The cells were trypsinized, and the number of viable cells was counted.

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