<i>Fli-1</i> pre-T LBL cells demonstrate elevated levels of intracellular NOTCH1 protein and carry <i>Notch1</i> mutations that result in ligand-independent transcriptional activation.

<p>A. <i>Fli-1</i> thymus spleen and bone marrow examined by flow cytometry for intracellular NOTCH1. Data is representative of three independent experiments. B. Intracellular NOTCH1 expression was analysed by flow cytometry in <i>Fli-1</i> preleukaemic thymi in comparison to the MigR1 control 6 weeks post transplant. Data is representative of three independent experiments. C. <i>Fli-1</i> pre-T LBL possess 5′ <i>Notch1</i> deletions detectable by PCR. Primers flanking the 5′ <i>Notch1</i> deletion site generate a small 485 bp PCR fragment of genomic DNA in <i>Fli-1</i> tumours, which is absent in control cells where the same primers span a 12,251 bp region of undeleted WT DNA. Lanes 1, 8 &amp; 21∶2-Log DNA ladder, 2–7 and 9–17: MigR1 control or primary and secondary (2°) <i>Fli-1</i> spleen cells (Spl), bone marrow (BM) or thymocytes (Thy) as indicated. 17; radiation-induced thymic lymphoma (RITL), 18: <i>Fli-1</i> transduced T cell precursors grown on OP9-DL1 cells, 19: <i>Fli-1</i>-transduced FDC-P1 cells, 20: <i>Mixl1</i>-transduced leukaemic BM cells. G3PDH: positive gDNA PCR control. D. <i>Notch1</i> mRNA upregulation in leukaemic <i>Fli-1</i> mice. Q-PCR of MigR1 control (Mig), <i>Fli-1</i> primary, secondary (2°) and <i>in vitro Fli-1</i> pre-T LBL cells from the thymus, spleen or liver. Samples and analysis as in Fig. 5. E. <i>Notch1</i> mRNA upregulation in preleukaemic <i>Fli-1</i> mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062346#pone-0062346-g001" target="_blank">Fig.1</a> F and 6B). F. Absence of 5′ <i>Notch1</i> deletions in preleukaemic <i>Fli-1</i> mice. Lane 1 &amp; 9∶2-Log ladder, lane 2: positive 5′ <i>Notch1</i> deletion control, 3–8: MigR1 control and preleukaemic <i>Fli-1</i> thymocytes as indicated.