Clonogenic Assay

HT-29 cells were seeded in T25 or 6-well plates (VWR, United States) in multiple cell concentrations, increasing with drug and radiation dose (0 Gy: 250–750, 2 Gy: 1,000–2,500, 4 Gy: 2,500–5,000, 6 Gy: 5,000–10,000, 8 Gy: 10,000–12,000, Sorafenib: 500–1,800 cells/well). After 24 h, cells were treated with 0, 100, or 250 nM seeMet 12 alone or in combination with other treatments (2 μg/ml sorafenib, 5 μg/ml sorafenib or radiation (0, 2, 4, 6, 8 Gy) and incubated at 37°C in an atmosphere containing 5% CO₂. After about 15 days, the medium was discarded, cells were washed with PBS and fixed using 95% ethanol for 10–20 min. Colonies were stained with hematoxylin (Histolab, Sweden) for 20 min, put in the water bath with tap water for 30 min, and let dry overnight. Colonies (defined as cell clusters that consist of >50 cells) counted. Plating efficiency (PE) and survival fraction (SF) were calculated as follows: PE = (number of colonies formed)/(number of cells seeded) × 100% and SF = (number of colonies formed after treatment)/(number of cells seeded × PE). Irradiation of the seeded cells was performed using a X-Rad 225 IR irradiator (Precision X-Ray Inc., Germany) with a 0.3 mm Cu filter, rotating table and a dose rate of 1 Gy/min. Experiments were repeated 4 times.