Ibl 637

In vitro IR was performed as described previously (12 (link)). Briefly, cells were treated with γ-rays (0.662 MeV) delivered using a ¹³⁷Cs laboratory irradiator (IBL 637, Cisbio) at a dose rate of 0.81 Gy/min.

Two days after reverse or standard transfection, the HaCaT cells were irradiated with 2 Gy from a ¹³⁷Cs source (IBL 637, CisBio International, Saclay, France) at a dose rate of 1.724 Gy/min or the Anémone/Bio irradiator (60Co, 2 Gy/min) in the ARC-Nucléart facility at CEA-Grenoble. After irradiation, the cultures were returned to the incubator. Thirty minutes later, the reverse-transfected cells were fixed in ice-cold methanol for 1 min and then washed three times with phosphate-buffered saline and stored at 4 °C. Standard transfected cells were lysed after 30 min of incubation using RIPA buffer (50 mM Tris–HCl, pH 7.4, 1 % Nonidet P-40, 0.25 % sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄, and 1 mM NaF) supplemented with 1× protease inhibitors (complete EDTA-free, Roche, Indianapolis, IN) to extract total proteins.

HeLa cells were pre-treated with DMSO, 1 or 2 for 20 h and irradiated using a ¹³⁷Cs γ-irradiator (IBL637, CIS Bio Int.; dose rate = 0.809 Gy min⁻¹). Solutions were removed 4 h after irradiation; cells detached using Trypsin and re-seeded in 6 well plates at a density of 1000 cells/well (in triplicate). Cells were incubated for 7–10 days after re-seeding to allow colony formation before being fixed with 10% methanol, 10% acetic acid and stained with 0.4% methylene blue. Colonies containing 50 cells or greater were counted using a Gelcount instrument and accompanying software (Oxford Optronix). Plating efficiencies were determined for each treatment condition and normalised to controls to provide the survival fraction. Resultant survival fraction (S.F.) versus radiation dose curves were fitted using a second order polynomial function (R² values > 0.99).
All other experimental details are described in the Supplementary Information.