Retroviral Transduction Assay for Rat-1 Fibroblasts

Retroviral transduction of Rat-1 fibroblasts with human EVI1 was carried out as described previously (28 (link)), using FUGENHD (Promega)- transfected packaging Plat-E cells (MSCV-EVI1-IRES-GFP, MSCV-EVI1-AQA-IRES-GFP or empty vector control MSCV-IRES-GFP). After 4 days cells were FACS-sorted by GFP, and equal levels of EVI1 expression were confirmed by western blot (Supplementary Figure S5A–C). GFP⁺ cells (10⁴) were seeded in untreated methylcellulose medium (MethoCult™ M3231, Stem Cell Technology), or supplemented with 30 μM of H₂O₂. Alternatively, 1 × 10⁵ cells in 100 μl (96-well plate) were left untreated or irradiated (0.5 or 2 Gy). After 14 days, colony number and size were quantified and documented using a DMIL inverted microscope fitted with a MC170 HD camera (Leica) (Supplementary Figure S5D). Induction of DNA damage was assessed by induction of γH2AX foci (Supplementary Figure S5E).