Plasmid midiprep kits

The propagated synthetic constructed vectors from E.coli bacteria were extracted using Plasmid Midiprep kits (Promega, Madison, USA). The cells were transfected with specific siRNA or synthetic vectors using Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

T24 and 5637 cells were transiently transfected with specific siRNA oligonucleotides, PVT1 siRNA, according to a previous study [28 (link)]. We chose the sequence, ‘CAGCCATCATGATGGTACT’, for further study. Non-specific siRNA (si-NC) and si-PVT1 were purchased from GenePharma, Suzhou, China. Bladder cancer cells were seeded in six-well plates and get 30–50% confluence before transfection. Cells were transiently transfected with si-PVT1 (100 nM) and si-NC (100 nM) by using Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Plasmid Midiprep kits (Promega, Madison, USA) were used to get the plasmid vectors (PVT1 shRNA, NC shRNA) for transfection.

Bladder cancer cells were transiently transfected with specific siRNA oligonucleotides and tetracycline-inducible double shRNAs vectors using Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. We chose the sequence, ‘TTAACCTCTTCCTATCTCA’ for further study [12 (link)]. Non-specific siRNA (si-NC) and si-CCAT2 were purchased from GenePharma, Suzhou, China. Plasmid Midiprep kits (Promega, Madison, USA) were utilized to get the plasmid vectors (CCAT2 shRNA, NC shRNA) before transfection.