Cells were trypsinized to create single cell suspensions, seeded into T-25 flasks at defined densities, and incubated overnight to ensure log phase of growth. The next day, two hours pre-irradiation, cells were fed with fresh media supplemented with either lapatinib (5 μM), U0126 (5 μM), or LY294002 (10 μM). Cells treated with nelfinavir (1 or 5 μM) received 2 or 26 h of pre-treatment prior to irradiation. Control cells were maintained in media containing a corresponding concentration of vehicle (DMSO or ethanol) alone. Cells were irradiated with single doses of 1, 3, 5, or 7 Gy using a Mark I 137Cs irradiator (JL Shepherd, San Fernando, CA) delivering a dose rate of 158 cGy/min. Two hours post-irradiation, all drugs were removed and the cells re-fed with fresh media. After 10 to 15 days, surviving colonies were fixed with a solution of methanol and acetic acid (3:1 v/v) and stained with 1% crystal violet. Colonies containing more than 50 cells were counted and survival curves were generated.
The surviving fraction was calculated from the number of colonies formed in the irradiated dishes compared with the number formed in the unirradiated control, where plating efficiency is defined as the percentage of cells plated that form colonies in unirradiated dishes, and surviving fraction = number of colonies formed/(number of cells plated × plating efficiency). Statistical comparisons were done using GraphPad Prism according to the two-tailed nonparametric Mann-Whitney test. The clonogenic survival curve for each condition was fitted to a linear-quadratic model (Y=e−[A * X + B * X2]) using GraphPad Prism according to a least squares fit, weighted to minimize the relative distances squared, and compared using the extra sum-of-squares F test. Each point represents the mean surviving fraction calculated from three independent experiments done in triplicate for each treatment condition; error bars represent the standard deviation. The mean inactivation dose was calculated according to the method of Fertil (21 (link)) and the cell survival enhancement ratio (ER) was calculated as the ratio of the mean inactivation dose under control conditions divided by the mean inactivation dose after drug exposure as described by Morgan (22 (link)). A value significantly greater than 1 indicates radiosensitization. For the drug dose response comparison, two-way ANOVA followed by Bonferroni posttests was performed using GraphPad Prism.
The surviving fraction was calculated from the number of colonies formed in the irradiated dishes compared with the number formed in the unirradiated control, where plating efficiency is defined as the percentage of cells plated that form colonies in unirradiated dishes, and surviving fraction = number of colonies formed/(number of cells plated × plating efficiency). Statistical comparisons were done using GraphPad Prism according to the two-tailed nonparametric Mann-Whitney test. The clonogenic survival curve for each condition was fitted to a linear-quadratic model (Y=e−[A * X + B * X2]) using GraphPad Prism according to a least squares fit, weighted to minimize the relative distances squared, and compared using the extra sum-of-squares F test. Each point represents the mean surviving fraction calculated from three independent experiments done in triplicate for each treatment condition; error bars represent the standard deviation. The mean inactivation dose was calculated according to the method of Fertil (21 (link)) and the cell survival enhancement ratio (ER) was calculated as the ratio of the mean inactivation dose under control conditions divided by the mean inactivation dose after drug exposure as described by Morgan (22 (link)). A value significantly greater than 1 indicates radiosensitization. For the drug dose response comparison, two-way ANOVA followed by Bonferroni posttests was performed using GraphPad Prism.
