All oligo DNAs and primers used are shown in Table S1. A BsaI-linker was ligated into pENTR/U6 (Thermo Fisher Scientific), resulting in pENTR/U6-Bgl2. The pAmCyan1-C1 plasmid (Takara Bio, Shiga, Japan) was digested with BglII and BamHI and then self-ligated to remove the multi-cloning site, resulting in pAmCyan1-noMCS. The AmCyan1-expressing cassette was amplified by KOD-neo-plus (Toyobo, Osaka, Japan) with AmCyan primers. Following BamHI digestion, it was inserted into the BglII site of pENTR/U6-Bgl2, resulting in pENTR/U6-AmCyan1. After BsaI digestion of pENTR/U6-AmCyan1, non-targeting (NT) short hairpin RNAs (shRNAs) (shNT), NEAT1-targeting shRNAs (shNEAT1a/b), or GABARAP-targeting shRNAs (shGBRPa/b) were ligated with Ligation High ver.2 (Toyobo). The shRNA and AmCyan1-expressing cassettes were transferred by LR reaction to pAd/BLOCK-iT-DEST (Thermo Fisher Scientific). Adenovirus vectors were constructed by transfection of adenovirus plasmid DNAs into 293T cells with LipofectAMINE2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol. The adenovirus titer was determined using the infectious genome titration protocol [55 (link)].
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