Anti-tumor effects of ANT2 shRNA and TRAIL in a breast cancer xenograft model in vivo. (A) Experimental protocol for tumor challenge and shRNA and TRAIL treatment. BALB/c nude mice were challenged with 5 × 106 MCF7 cells by subcutaneous injection into the right flanks. After 21 days, they began 3 days of treatment with thrice daily intraperitoneal injections of PBS or human recombinant TRAIL with or without thrice daily intratumoral injections of Lipofectamine 2000-supplemented scrambled or ANT2 shRNA vector. Calipers were used to make weekly measurements of tumor dimensions, and tumor volumes were calculated as described in the Methods. Measurements continued until day 45 after tumor challenge. Data were analyzed using the Student t test. Differences are considered statistically significant at P < 0.05. (B) Expression of TRAIL receptors in tumors after shRNA and TRAIL treatment. Tumors were isolated on day 45 after tumor challenge and subjected to RT-PCR using primers specific for human DR4, DR5, DcR2, and GAPDH (internal control).