*4.5. Clonogenic Assay*

Cells were seeded in 50 mm cell culture dishes at a very low density (1000 cells for treatments and less (500 or 250) for the controls). After the attachment, cell cultures were treated for 45 min with BLEO in the absence or presence of inhibitors. Then, the culture media was changed and fresh inhibitors were added. The cells were incubated until control colonies reached at least 50 cells (counted under the microscope) for 8 days. Finally, colonies were fixed on 70% ethanol at room temperature (RT) for 10 min, briefly stained with 0.1% crystal violet (Fluka 61135), rinsed in abundant distilled water, dried at 37 ◦C, and manually counted by an observer under a magnifying glass. The plates were blind-coded. The clonogenic efficiency was expressed relative to the controls.
