Culture and maintenance of cells

Immortalized MEFs and fibroblasts were cultured in standard conditions, at 37°C and 5% CO₂. The cell culture medium was composed of Dulbecco’s modified Eagle’s medium [glucose (4.5 g/liter), GlutaMAX, and sodium pyruvate; Gibco Life Technologies] supplemented with 10% “Fetal Bovine Serum Premium, South American Origin” (Biowest) and penicillin-streptomycin (Pen-Strep) (Gibco Life Technologies). In conditions of mitochondrial dysfunction (induced either genetically or by treatment), the medium was additionally supplemented with uridine (50 μg/ml). At 90% confluency, cells were split cell type–dependently in ratios ranging from 1:4 to 1:20.

Embryos from embryonic day 13.5 of intercrossed CHOP KO (Chop^−/−) mice were used to isolate primary MEFs (59). Immortalization was achieved by transformation with the SV40 T antigen.

For induction of mitochondrial dysfunction by actinonin treatment, 80% confluent cells were treated for 48 hours with 100 μM actinonin (Sigma-Aldrich). Proteasome was inhibited with 15 μM MG132 for the last 6 to 8 hours of treatment as indicated. Inhibition of the ISR was achieved by 4- or 48-hour 1 μM ISRIB (Sigma-Aldrich) treatments of 90% confluent cells. All compounds were solubilized in dimethyl sulfoxide (DMSO). Untreated cells were supplemented with corresponding amounts of the solvent. Treatments were renewed on a daily basis.

Transfection of plasmids conferring hygromycin resistance (pTK-Hyg LIP, pTK-Hyg LIP^western, pTK-Hyg LAP, and pTK-Hyg C/EBPβ) was performed with Lipofectamine 2000 or Lipofectamine LTX (Invitrogen) according to the manufacturer’s instructions using the forward transfection procedure. Seventy-two hours after transfection, the culture medium was replaced by hygromycin-supplemented (100 μg/ml) medium for negative selection of untransfected cells. Transfected cells were maintained in hygromycin-supplemented (100 μg/ml) medium.

To estimate differences in cell growth caused by CHOP deficiency and/or mitochondrial dysfunction, an equal number of cells were seeded and treated as indicated. The numbers of cells were determined at the indicated time points using the Countess Automatic Cell Counter (Invitrogen) combined with trypan blue staining.