MYCN promotes ETP population growth while inhibiting Notch-induced T-cell differentiation. (A) qRT-PCR analysis for Mycn and Myc expression in RNA harvested from sorted ETPs from Rosa26-CreER^T2 control or Rosa26-CreER^T2Zmiz1^f/f mice that had been injected with tamoxifen 40–44 h prior. n = 5/group. (B)Mycn qRT-PCR analysis of control vector and Mycn-transduced ETPs (separated into GFP^low and GFP^high subsets) compared with human ETP-ALL (LOUCY; CUTLL3) using primers that detect both mouse and human Mycn generated on OP9-DL1 stroma as in Fig. 4 A; n = 3/group. (C) qRT-PCR analysis of the sorted cells in B for Notch ETP target genes Tcf7, Gata3, and Hes1; n = 3/group. (D–L) Additional analysis of the experiment in Fig. 4 A showing the separation of cells in the four indicated conditions based on GFP^low and GFP^high expression (D–G). %ETP (H and J) and %DN2 (I and K) were measured in GFP^low (H and I) and GFP^high (J and K) subpopulations as indicated in D–G and used to calculate a “ETP-to-DN2 inhibition index” based on the indicated ratio that measures how strongly ectopic MYCN increases the ETP/DN2 ratio relative to vector control (L); n = 3/group. Unless noted otherwise, 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.