Pad block it dest

Adenoviral shRNA constructs were obtained by synthesizing the respective oligonucleotide sequences into pENTR-U6 followed by homologous recombination into the pAd-BlockIt-DEST (all Invitrogen) according to the manufacturer’s instructions. The final constructs were linearized with PacI and purified. After transfection in HEK293A cells using Lipofectamine 2000, virus was harvested 10 days post infection. Crude virus lysate was used to re-infect further HEK293A cells and final harvested virus was purified by column purification (Virapur). For adenoviral over-expression viruses, the respective genes were synthesized (HiGene Molecular Biotech) with a C-terminal HA-tag and cloned into pENTR_CMV (M. Montminy) with HindIII and XhoI. They were recombined into the same pAdBlockIT-DEST destination vector to obtain comparable transduction rates. Concentration of active virus particles was determined by a plaque assay as recommended by the virus purification manual. For viral transduction of preadipocytes, 1.25 · 106 pfu of virus in 50 μl OptiMEM with 1% Lipofectamine2000 were incubated for 30 min. The mixture was combined with 50 μl of cells at 12,500 cells/μl and added to a 96well. 4 h post transduction virus-containing medium was replaced by full growth medium.

Adenovirus vectors expressing nontargeting (NT) short hairpin RNA (shRNA; shNT), NEAT1-targeting shRNAs (shNEAT1a/b), and GABARAP-targeting shRNAs (shGBRPa/b) have been reported previously [10 (link)].
Adenovirus vectors expressing SOD2-targeting shRNAs (shSOD2a/b) were constructed as reported previously [8 (link)]. Briefly, oligo DNAs (Table S1) were ligated into BsaI-digested pENTR/U6-AmCyan1 with Ligation High version 2 (Toyobo, Osaka, Japan). Then, shRNA and AmCyan1-expressing cassettes were transferred by the LR reaction to pAd/BLOCK-iT-DEST (Thermo Fisher Scientific, Waltham, MA, USA). Adenovirus vectors were constructed by transfection of PacI-digested adenovirus plasmid DNA with LipofectAMINE2000 into 293A cells (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Adenovirus titer was determined by the infectious genome titration protocol [63 (link)]. When knocking down genes in irradiated cells, these adenoviruses were transduced immediately after irradiation.