Mrc 5

Fibroblast cell lines include: BJ-5ta (ATCC, CRL-4001), BJ1-hTERT (kind gift from Vilhelm A. Bohr, Laboratory of Molecular Genetics, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD), MRC-5 (kind gift from Tim Sparer, University of Tennessee, Knoxville, Knoxville, TN), AG04405 (ATM-hTERT) (kind gift from Peter Lansdorp, University of British Columbia, Vancouver, Canada), and GM02052 (Coriell). The lymphoblastoid cell line GM12878 (Coriell) was also used. Information about cell lines and media formulations is summarized in Supplementary Table 2. All growth media and fetal bovine serum was purchased from Corning, except for GM12878 growth media from Gibco. All fibroblast lines were passaged at a density of 80%. For irradiation experiments, fibroblast lines were grown to confluency prior to X-ray exposure. For the GM12878 suspension cell line, cells were passaged at a density around 500,000 cells per 1 mL of medium. For irradiation experiments, GM12878 cells were grown to a density of 1 million cells per 1 mL of medium prior to X-ray exposure.

Fibroblast cell strains of three genetic backgrounds and two lineages were used in this study: normal human dermal fibroblasts (NHDF; Heidelburg, Germany), human diploid foetal lung fibroblasts (MRC-5; Coriell Institute for Medical Research) and neonatal foreskin fibroblasts (HF043; Dundee Cell Products, UK). Standard culture conditions were a seeding density of 6 × 10⁴ cells/cm² in media (C-23020, Promocell, Heidelburg, Germany) containing 1% penicillin and streptomycin, and a fibroblast-specific supplement mix consisting of foetal calf serum (3% v/v), recombinant fibroblast growth factor (1 ng/ml) and recombinant human insulin (5 μg/ml) (Promocell, Heidelburg, Germany). For the assays requiring senescent cultures, cells were counted and equal numbers of cells seeded at each passage until the growth of the culture slowed to less than 0.5 PD/week as previously described [2 (link)] (this occurred at PD = 64 (NHDF), 65 (MRC-5) and 64 (HF043). Viable cell numbers were determined at each passage by trypan blue staining. For cultures grown under serum starvation conditions, cells were maintained in DMEM (Sigma Aldrich, Dorset, UK) supplemented with 0.1% of serum and 1% penicillin and streptomycin in the absence of fibroblast-specific supplement, for 24 h prior to treatment.