Genjet version 2

TRExBCBL-1-RTA (a gift from Dr. Jae U. Jung, herein termed iBCBL-1) and its derivative lines were cultured in RPMI 1640 medium (Quality Biological) supplemented with 15% heat-inactivated fetal bovine serum (FBS), stable L-alanyl-glutamine (Glutamine XL), and streptomycin and penicillin, at 37 °C and 5% CO₂. 293T, HeLa.Kyoto (a gift from Dr. Ron R. Kopito), and their derivative cell lines were cultured in DMEM supplemented with 10% FBS and antibiotics. The cell lines were tested for mycoplasma contamination (R&D systems) and if necessary cultured in plasmocin™ treatment or prophylactic (InvivoGen). Transient transfection with plasmids was performed using GenJet version II (SignaGen Laboratories). For stable and doxycycline (Dox)-inducible expression of short hairpin RNAs (shRNAs), culture cells were lentivirally transduced with shRNA-specifying sequences in the presence of 10 µg/ml polybrene overnight, and stably transduced cells were selected by growing in the presence of 1 µg/ml puromycin or 400 µg/ml geneticin for more than 1 month, and pooled clones were collected. For detection of mitochondrial superoxide, cells were incubated with 5 µM MitoSOX red indicator (Invitrogen) in Hank’s buffered salt solution containing calcium and magnesium for 10 min at 37°C just before cell fixation.