Ecm0749l

As target cells for assessment of malignant transformation, we used hTERT-HPNE E6/E7/K-RasG12D (CRL-4038, ATCC, Manassas, VA, USA) and HEK293T XPack CMV-XP-GFP-EF1Puro (XPAK530CL-1, System Biosciences, Palo Alto, CA, USA). The hTERT-HPNE cell line represents the intermediary stage during acinar-to-ductal metaplasia in pancreas. They have an undifferentiated phenotype and active Notch signalling pathway. The HEK293T cell line is a highly transfectable derivative of human embryonic kidney 293 cells and contains the SV40 T-antigen. Both hTERT-HPNE and HEK293T cell lines were maintained in Dulbecco’s Modified Eagle Medium (ECM0749L, EuroClone, Milan, IT) supplemented with 10% of either PDAC patients’ serum or healthy donor serum (without FBS), 1 mM L-glutamine (BE17, 605E, Lonza, Verviers, BE), 1 mM penicillin-streptomycin (ECB3001D, EuroClone), and 1% of MEM non-essential amino acids for HEK293T cell line.

Hep3B and HepG2 cell lines were purchased from the Leibniz-Institut DSMZ, Braunschweig (ACC-93, ACC-180) and grown in DMEM (Euroclone, ECM0749L) and RPM1640 (Euroclone, ECB9006L), respectively, supplemented with 10% FBS (Euroclone, ECS0180L) and 1% penicillin-streptomycin. HuS cells were a gift of Dr. Vinicio Carloni, University of Florence and were grown in DMEM containing 4.5 g/L of glucose (Euroclone, ECB7501L) and supplemented with 10% FBS (Euroclone, ECS0180L), 5 ng/mL EGF (Sigma-Aldrich, E9644), 420 ng/mL insulin (Sigma-Aldrich, I9278), 20 ng/mL selenium (Sigma-Aldrich, S9133), 1% DMSO (Amresco, 0231) and 1% penicillin-streptomycin (Euroclone, ECB3001D). Cells have been authenticated by STR PCR by the supplier and routinely tested for Mycoplasma contamination using Myco Alert (Lonza, LT07-318).

Osteosarcoma 143B Tk⁻ cells and colorectal cancer HCT116 cells were used, both carrying wild-type mtDNA. 143B cells were purchased from ATCC (#CRL-8303) and HCT116 cells were a kind gift from Prof. Paolo Pinton from the University of Ferrara. Cell origin was authenticated using AMPFISTRIdentifiler kit (Applied Biosystems #4322288) and their STR profile corresponded to their putative background (Supplementary Fig. 1a). For basal conditions, cells were cultivated in Dulbecco’s modified Eagle medium (DMEM) high glucose (Euroclone #ECM0749L), supplemented with 10% FBS (Euroclone #ECS0180L), L-glutamine (2 mM, Euroclone #ECB3000D), penicillin/streptomycin (1 ×, Euroclone #ECB3001D), and uridine (50 µg mL⁻¹, Sigma-Aldrich #U3003), in an incubator with a humidified atmosphere at 5% CO₂ and 37 °C. Cells were replaced by a fresh batch after 15 passages and mycoplasma testing was performed before disposal and after each thawing (approximately every 2 months). Experiments in hypoxia were performed using an Invivo2 300 (Baker Ruskinn) chamber, set at 5% CO₂, 37 °C and 1% O₂. Where indicated, cells were incubated for 3 h with dimethyloxalylglycine [DMOG (1 mM), Sigma-Aldrich #D3695] or MG132 (10 μM, Sigma-Aldrich #M7449) and 1 h with pimonidazole (100 μM, Hypoxiprobe #70132-50-3).