Clonogenic cell survival assay

After treatment (24 h exposure), cells were trypsinized, counted, and 800 cells per well were re-plated in six-well plates for colony formation assays. After incubation for 10–14 days, colonies were fixed with methanol:acetic acid (3:1; pre-cooled at 4°C) at 4°C for 10 min, and stained with 1% crystal violet at room temperature for 15 min, and then counted. A light microscope (Nikon Corporation) was used to observe and count colonies, and GraphPad Prism 6.0 software (GraphPad Software, Inc.) was used for quantification and analysis. Plating efficiency (PE) was determined, and the surviving fraction was calculated based on the number of colonies that arose after treatment and is expressed in terms of PE. Each experiment was repeated three times.