Clonogenic assay

Cells were seeded in 25 cm² flasks at 10⁵ cells/flask. When cultures were in exponential growth phase, medium was removed and replaced with fresh medium (including 1% or 10% serum) containing drug. For radiation treatment, cells were irradiated using an X-Strahl RS225 x-ray irradiator (Xstrahl Limited, Surrey, United Kingdom) at a dose rate of 1.6 Gy per minute. Cells were then incubated for 24 h at 37ºC in 5% CO₂. After treatment, cells were counted and seeded for clonogenic survival assay as previously described.15 Cells were incubated at 37°C in 5% CO₂ for up to 13 days. LNCaP cells did not form clonogens under similar conditions and so were not used for clonogenic assay. Colonies were fixed in methanol, stained with crystal violet solution, and colonies of at least 50 cells were counted.