Exogenous Ku70 and Ku80 overexpression

The full-length human Ku70 cDNA and Ku80 cDNA were provided by Dr. Xiang at Shanghai Jiao Tong University School of Medicine [25], individually sub-cloned into pSuper-flag-puro construct. The construct and the adeno-associated virus (AAV) package plasmids (Genechem) were co-transfected to HEK293 cells to generate Ku70- or Ku80-expressing AAV [36]. At a density of 3 ×10 ⁵ cells/well THP-1 cells were seeded into six-well plates. The virus was enriched, filtered and added to THP-1 cells. Afterwards, cells were cultured in fresh complete medium. Puromycin (5.0 μg/mL) was added to select resistant stable cells for four more passages. Expression of Ku70 and Ku80 was verified by qPCR and Western blotting. Control cells were transfected with AAV with empty vector.