Mouse embryonic fibroblasts (MEFs) from a C6ko mouse and its C6wt littermate were generated from day 12.5 embryos resulting from timed-pregnant heterozygous breedings and tissues not used for culture were genotyped. The generation and characterization of C6ko mice is described elsewhere [27] . Single pups were dissected in ice cold PBS, the body without head, limbs and liver, lung and heart was minced, the centrifuged pellet was digested with 0.25% Trypsin-EDTA at 37uC for 15 min and neutralized with MEF medium (Dulbecco's modified Eagle medium with high glucose, 10% fetal calf serum, 2 mM L-Glutamine, 100 mM non-essential amino acids, 1 mM sodium pyruvate, 1 mM b-mercaptoethanol). Cells were passaged through a pipette tip, centrifuged and suspended in MEF medium containing DNase I. After centrifugation, cells were suspended in fresh medium, incubated for 2 min at room temperature to let debris and cell clumps settle and cells in the supernatant were seeded into cell culture flasks. At passage 2, cells were immortalized by transfection with pSV3-neo SV40 large T antigen (ATCC) with the Fugene reagent (Roche) according to manufacturer's instructions. Immortalized cells were selected and propagated through the addition of 600 mg/ml G418 to the medium.