Figure 3.

Activating Notch1 mutations and high NOTCH1 protein levels in SCL–LMO1 T-ALL are dependent on Cd3 gene status. (A) The Notch1 gene from Cd3ɛ^+/+SCL^tgLMO1^tg and Cd3ɛ^−/− SCL^tgLMO1^tg tumors was sequenced in three overlapping amplicons. Shown are the positions of the primers used in the sequencing reactions. HD, TAD, and PEST represent the heterodimerization, transactivation, and protein degradation domains, respectively. (B) NOTCH1 intracellular staining in Cd3ɛ^+/+SCL^tgLMO1^tg and Cd3ɛ^−/−SCL^tgLMO1^tg primary tumor samples. The average fluorescence intensity per pixel from Z-stack confocal analysis from two Cd3ɛ^+/+SCL^tgLMO1^tg and three Cd3ɛ^−/−SCL^tgLMO1^tg different tumor samples is shown as mean ± SD. (C) Quantitative RT–PCR of the indicated NOTCH1 target genes were performed in Cd3ɛ^+/+SCL^tgLMO1^tg and Cd3ɛ^−/−SCL^tgLMO1^tg tumors. Relative mRNA expression levels in tumor cells are normalized by CT values obtained with Cd3ɛ^+/+SCL^tgLMO1^tg cells and are presented as box plots with the median and extreme values of each distribution ([*] P < 0.05; [**] P < 0.01; [***] P < 0.001 as compared with Cd3ɛ^−/−SCL^tgLMO1^tg tumors). (D) The Notch1 gene from purified late preleukemic and leukemic thymocyte subsets from Cd3ɛ^+/+ SCL^tgLMO1^tg mice was sequenced as in A. (ISP8 corresponds to Thy1.2⁺CD8⁺CD4⁻Cd3ɛ^lo, SP4 corresponds to Thy1.2⁺CD4⁺CD8⁻, and SP8 corresponds to Thy1.2⁺CD4⁻CD8⁺.)