Clonogenic cell survival assay

The clonogenic survival assay was used to evaluate the radiosensitivity of the TE1, TE8, and TE11 cells infected with hdm-2siRNA. Briefly, the transfected cells were cultured in six-well plates with an appropriate cell density, allowed 24 h to attach and subsequently exposed to 60-Co γ-rays. After irradiation, the cells with renewed media were incubated for 14 days at 37°C in a humidified incubator. The produced colonies were counted after fixing with a mixture of methanol-acetic acid (3:1) followed by staining with 2% Giemsa (Merck, Germany). The colonies consisting of more than 50 cells were scored as the survivors. Then plating efficiency and survival fraction were calculated and the sensitizing enhancement ratio was defined as the radiation was required to obtain a specific surviving fraction (SF). Each data point was performed in triplicate. The obtained data were normalized to the plating efficiencies of the sham treated control and used to plot the survival curves by means of the GraphPad Prism 6 (GraphPad Software, Inc., San Diego, CA, USA).