Silencing of c-Myc prolongs survival and reduces LIC frequency in mice transplanted with murine T-ALL cells. (A) Experimental design. Five independent murine Tal1/Lmo2 T-ALL cells were infected with retroviruses encoding an shRNA to c-Myc or Renilla luciferase. Cells were sorted for GFP expression, serially diluted, and transplanted into syngeneic recipients via intraperitoneal injection. Transplanted mice were monitored for evidence of disease. (B) The survival curve for each group of mice was estimated using the Kaplan-Meier method and the difference in overall survival between the 2 groups assessed by the log-rank test (P < .0001). (C) c-Myc silencing reduces LIC frequency in mice transplanted with murine T-ALL cells. A log-log plot and LIC frequency was calculated using ELDA software for ShRenilla (red, 1/7308) and shMyc (black, 1/41 337). A portion of secondary recipients develop leukemia when transplanted with limiting dilutions of shRenilla- and shMyc-infected leukemic cells. (D) Reduced c-Myc protein levels in shMyc-transduced leukemic cells at time of transplant. Protein was isolated from GFP-positive leukemic cells prior to transplant and lysates probed with a c-Myc and Erk1/2 antibodies. (E) Transplanted mice that develop disease do not exhibit reduced Myc expression. RNA was isolated from leukemic cells transduced with the shRen or shMyc retroviruses and c-Myc mRNA levels determined by quantitative real-time PCR. Copy number was normalized to β-actin using the ΔΔCT method. Each point represents c-Myc mRNA levels of leukemic cells isolated from a single mouse.