4.6. Transfection and Stable Cell Lines

The shRNA plasmids used in this study were constructed using the pGPU6/GFP/Neo plasmid (GenePharma, Shanghai, China). The targeting sequences were 5′-GCA TCA CGC TTG ACC CAT T-3′ (sh-IGFL2-AS1) for the human IGFL2-AS1 transcript and 5′-TTC TCC GAA CGT GTC ACG T-3′ (sh-NC) for the non-targeting control [8]. Plasmids were transiently transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, after which stably expressing cells were selected by culturing in the presence of the neomycin analog, G418. Cells were incubated for 14 days, with the replacement of G418-containing medium every 3 days. After 2 weeks, green fluorescent protein (GFP)-positive clones were isolated and maintained in the presence of G418. Fluorescence images were obtained using an Eclipse Ti fluorescence microscope (Nikon, Tokyo, Japan).