Geneticin

Restriction enzymes and T4 DNA ligase were acquired from Fermentas - Thermo Fisher Scientific, Pittsburgh, PA, USA. Antibiotics hygromycin (HG), phleomycin (Phleo), and geneticin (G418) were acquired from InvivoGen (San Diego, CA, USA). pYL16 and nourseothricin (nour) were acquired from Werner BioAgents (Jena, Germany) using concentrations recommended by the supplier for selection of the respective antibiotic resistance marker-bearing S. cerevisiae transformants. Ampicillin and kanamycin were acquired from Sigma-Aldrich (Zwijndrecht, The Netherlands). Oligonucleotides used for strain constructions were purchased from Sigma-Aldrich. Yeast genomic DNA was isolated from yeast using the YeaStar™ Genomic DNA Kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s instructions.

All cell lines were cultured at 37 °C in a 5% CO₂ humidified atmosphere. U2OS cell lines stably expressing GFP, GFP-Ku80, RFP-53BP1 or FLAG-UCHL3 (WT, C95A, S75A and S75E) were cultured in Dulbecco’s modified Eagle medium (DMEM, Nacalai Tesque) containing 10% fetal bovine serum (FBS, Sigma-Aldrich), 100 U/ml penicillin (Nacalai Tesque), 100 μg/ml streptomycin (Nacalai Tesque), 584 μg/ml L-glutamine and 500 μg/ml Geneticin (Thermo Fisher Scientific). U2OS stably expressing the HR reporter direct repeat-GFP, EJ2-GFP reporter^{40 (link)} and U2OS UCHL3 knock out cell lines were cultured in identical media with 1 μg/ml puromycin (InvivoGen) instead of Geneticin.

HepG2 and H9c2 were purchased from American Type Culture Collection (ATCC). Human mesenchymal stem cell (hMSCs) was purchased from Gibco (Carlsbad, CA, USA). HepG2 and R-HepG2 were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin antibiotics mixture (PS), except that R-HepG2 cells were supplemented with 1.2 μM of DOX in culture medium. H9c2 and hMSCs were cultured in DMEM and alpha-MEM supplemented with 10% FBS and 1% PS, respectively. hMSCs were assayed within four passages. To generate stable cell lines constitutively expressing the mitochondrial-RFP tag for mitochondria visualization, a construct pDsRed2-Mito (Clontech, CA, USA) carrying a human cytochrome C oxidase subunit VIII mitochondrial targeting sequence was transfected to cells using lipofectamine-3000 reagent according to manufacturer’s protocol. After 48 h of incubation, untransfected cells were eliminated by 1 mg/mL of Geneticin (InvivoGen, San Diego, CA, USA). To ensure clonal purity, transfected cells were sorted to a single clone by a cell sorter (FACSMelody, BD Biosciences, San Jose, CA, USA). Cells carrying the mitochondrial-RFP tag were expanded from a single clone. Fluorescence of the RFP-labelled mitochondria was verified by flow cytometry (FACSVerse, BD Biosciences, San Jose, CA, USA) and confocal microscopy (SP8, Leica, Wetzlar, Germany).

NCI-H1975 WT cells were transfected with a myc-DDK-tagged FLJ23584 ORF clone (RC203355; Origene) using the SF Cell Line 4D-Nucleofector X Kit L (V4XC-2024; Lonza). The same plasmid was used for rescue experiments in non-targeting control and C22orf46 knockout cell lines. For each transfection, one million cells and 5 μg plasmid were used, and the manufacturer’s instructions were followed. 4 d after transfection, the selection with Geneticin (InvivoGen, ant-gn) was started. The cell populations resistant to 600 μg/ml Genetecin were subjected to downstream experiments.

A375 cells stably expressing the soluble version of human tyrosinase (A375-ST Tyr) were grown in DMEM (Gibco-31966–021) supplemented with 10% FBS (Gibco) and 400 μg/ml Geneticin (Invivogen) as previously described (Chiritoiu et al., 2016 (link)). Cells at confluence of 65%–70% were treated with 30 μM kifunensine (Santa Cruz Biotechnology) overnight (ON) or 20 μM MG132 (Santa Cruz Biotechnology) for 4 h and harvested accordingly.

For the generation of HFF and REF cells with doxycycline-inducible expression of humIE1 or ratIE1, replication-deficient lentiviruses were generated using pInducer20-based expression constructs. For this purpose, HEK293T cells seeded in 10 cm dishes (5 x 10⁶ cells/dish) were transfected with a pInducer20-based vector together with packaging plasmids pLP1, pLP2, and pLP/VSV-G using the Lipofectamine 2000 reagent (Invitrogen). Viral supernatants were harvested 48 h after transfection, clarified through a 0.45-μm filter and stored at -80°C. HFF or REF cells were incubated for 24 h with lentivirus supernatants in the presence of 7.5 μg/mL polybrene (Sigma-Aldrich). Stably transduced, IE1-expressing cell populations were selected by adding 500 μg/mL geneticin (Invivogen) to the cell culture medium containing 7 to 10% tetracycline-free fetal bovine serum (Clontech). IE1 expression was induced by addition of 500 ng/mL doxycycline (Sigma-Aldrich). To generate PML-knockdown and control cells, HFF were transduced with lentiviral supernatants produced from pLVX-shRNA-based expression constructs, followed by selection with 5 μg/mL puromycin (Invivogen).