TUNEL assay To determine apoptosis in tumor cells treated with T oligo and radiation, TUNEL staining was performed using ApopTag Plus peroxidase kit in a method previously described with some modification. To quantify the apoptotic cells, three to five high power fields per mouse were selected and apoptotic cells were counted by two investigators. Tumorigenesis method of cancer cells treated with T oligo and radiation To assess the growth potential of T oligo and IR treated tumor cells in mice, Inhibitors,Modulators,Libraries mammary tumor cells were supple mented in culture with T oligo for 24 hours, and then irradiated with 3 Gy. The tumor cells cultured Inhibitors,Modulators,Libraries in med ium alone or containing control oligo were used Inhibitors,Modulators,Libraries as con trol groups. After radiation, the cells were washed and prepared for injection.

Untreated cells were resuspended in PBS, while the control oligo and T oligo treated cells were resuspended in 1. 2 mM control oligo or T oligo in PBS. T oligo or control oligo treated and medium only treated tumor cells were injected respectively in the right or left flanks of syngeneic wild type mice. Untreated tumor cells with or without IR were also injected as controls. Inhibitors,Modulators,Libraries The mice were followed for up to 30 days after the tumor inoculation and tumor growth was measured with calipers every two days. The tumor incidence was also recorded. In vivo treatment MMT mice at approximately 73 days of age were injected intraductally in the right chest mammary gland with 50 uL of a T oligo solution. The left chest mammary gland in the same mouse was injected with the same dosage of con trol oligo.

After seven to eight daily injections, the mice were sedated with intraperitoneal administration of keta mine and xylazine and placed in a special Irradiation Pie cage. The mammary glands were irradiated with 3 Gy IR with the rest of the body covered by lead Inhibitors,Modulators,Libraries foil. The mice were sacrificed 10 days after the radiation. The treated mammary glands were then removed, whole mounted, formalin fixed, stained in carmine alum, and photographed with a digi tal camera. To quantify the tumor burden, digital images of all whole mounted mammary glands were analyzed using SPOT advanced software. Statistical analysis Statistical significance was determined using Students t tests or X2 test. One way ANOVA was used for analysis of data with more than two subgroups.

Results Sensitization of tumor cells to radiation by T oligo To determine if T oligo can enhance the inhibition of mammary tumor cell growth due to radiation, tumor cells from MMT mice were cultured with T oligo at concentrations ranging from 0 to 40 uM for 24 hours and then irradiated. Tumor cells cultured in DMEM medium alone or DMEM containing the same concen tration of Ceritinib supplier a control oligo were used as controls. The cells were collected and counted at 0, 24, 48, 72 and 96 hours after exposure to 3 Gy IR. T oligo alone inhibited growth of tumor cells in a dose dependent manner, with the most marked inhibition at 40 uM T oligo.