miR-146 overexpression delays c-myc upregulation associated with premalignancy. (A) Western blot analysis showing c-myc upregulation in total thymocytes from late premalignant 12-week-old tPTEN^−/− mice and both thymocytes and mature T cells from malignant 13.5-week-old tPTEN^−/− mice (C57BL/6 background). C-myc upregulation is not detected in the thymocytes or mature T cells of littermate 13.5-week-old 146a8/tPTEN^−/− mice. Data are representative of 2 independent experiments. (B) Quantitative RT-PCR analysis of c-myc expression in total thymocytes of 14- and 15-week-old mice. c-myc is upregulated in the thymocytes of tPTEN^−/− mice (with tumors), but not in thymocytes from tumor-free littermate 146a1/tPTEN^−/− or 146b/tPTEN^−/− mice. Data are presented as mean ± SD and are representative of 2 independent experiments per transgenic line. Statistics were calculated by Student t test (***P < .001, **P < .01). (C) Classification of littermate or age-matched 13.5- to 15-week-old tPTEN^−/− and 146/tPTEN^−/− mice based on the criteria outlined in Figure 3 (data from all transgenic lines were pooled). (D) Western blot analysis of c-myc protein expression in lymph node tumor cells of 12.5-week-old littermate tPTEN^−/− and 146a1/tPTEN^−/− mice. Data are representative of 3 independent experiments. (E) Fluorescence in-situ hybridization showing TCRα/c-myc chromosomal translocations in interphase-arrested lymph node tumor cells from the tPTEN^−/− and 146a1/tPTEN^−/− mice in (D). Images were captured at original magnification ×40.