To obtain cell lines stably expressing PCAT1, KYSE150 and KYSE450 cells were transfected with the pcDNA3.1 control plasmid or pcDNA3.1-PCAT1 plasmid using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions and then selected with G418 (300 μg/mL) for 2 weeks. To generate cell lines with stable knockdown of PCAT1, KYSE30 cells were transfected with the pGPH1/Neo control plasmid (sh-NC) or PCAT1-sh-1/sh-2 plasmid using Lipofectamine 2000 and selected with G418 (300 μg/mL) for 2 weeks. The cell lines with stable overexpression or knockdown of PCAT1 were identified using RT-qPCR.
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