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CCK-8 assay
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Cells were seeded in 96-well plates at a density of 3 × 10^3^/well and incubated for 24 h. The cells were then treated with 5 ng/ml TGF-β1 for 48 h and were incubated for 2 h with a CCK-8 solution (Dojindo, Kumamoto, Japan). The optical density (OD) of each well at 450 nm was measured with an ELISA plate reader (Spectramax M2, Molecular Devices, USA).
Apoptosis assay
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Cell apoptosis was evaluated using a commercial kit (BD Pharmingen, CA, USA) based on a protocol provided by the manufacturer. Stained cells were analyzed with a FACSCalibur flow cytometer (Becton Dickinson, CA, USA).
Colony formation assay
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Approximately 100 cells were plated per well in triplicate into six-well tissue culture plates and cultured in DMEM containing 10% FCS, which was replaced every 4 days. After 10 days of culture, the plates were stained with crystal violet and colonies were counted.
RNA isolation and qRT-PCR
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Total RNA was extracted from cells using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized from the RNA using a RevertAid First Strand cDNA Synthesis Kit
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