MYCN promotes ETP population growth and myeloid cell fate while inhibiting Notch1-induced T-cell differentiation. (A–F) Representative Kit/CD25 flow cytometry plots (A); ETP (Kit^hiCD25⁻, B), myeloid progenitor (Kit^loCD25⁻, C), and DN2 (D) absolute counts within the DAPI⁻ CD45⁺ GFP⁻ NGFR⁺ Lineage⁻ CD44⁺ gate; representative CD11b⁺Gr-1 flow cytometry plots (E); and CD11b⁺Gr-1⁺ (F) absolute counts after 7 days of culture in the OP9-DL1 assay described in Fig. 2 A of vector- or Mycn-transduced LMPPs treated with OHT from Rosa26-CreER^T2 control or Rosa26-CreER^T2Zmiz1^f/f mice. Cell counts are shown for 1,000 seeded NGFR⁺ LMPP cells; n = 3/group. (G) Experimental strategy of competitive transplant assay using CD45.2⁺Il7rCre or Il7rCre Mycn^f/f as donor mice. (H–L) %CD45.2⁺ chimerism of indicated thymocyte subsets. ETP = Lineage⁻CD44⁺CD25⁻cKit^hi; DN2a = Lineage⁻CD44⁺CD25⁺cKit^hi; DN2b = Lineage⁻CD44⁺CD25⁺cKit^lo; DN3 = Lineage⁻CD44⁻CD25⁺; DN4 = Lineage⁻CD44⁻CD25⁻; n = 10 (control) and 7 (knockout)/group. (M–O) Representative Kit/CD25 flow cytometry plots (M); absolute counts of ETP, DN2a, and DN2b cells within the CD45⁺ GFP⁻NGFR⁺Lineage⁻CD44⁺ gate (N); and qRT-PCR for Mycn of indicated sorted cells (O) after 7 days of OP9-DL1 culture described in Fig. 2 A. Cell counts are shown for 1,000 seeded LMPP cells; n = 3/group. P values were based on two-sided t tests for two samples and one-way ANOVA for more than two samples. *P < 0.05; **P < 0.01; ***P < 0.001.