ZMIZ1 promotes ETP population growth and inhibits Notch1-induced T-cell development. (A) Experimental strategy to show effects of retroviral transduction of ZMIZ1 on the generation of ETPs and Notch1-induced immature T cells from BM LMPPs using the OP9-DL1 culture assay. LMPPs = Lineage⁻Sca-1⁺Kit^hiFlt3^hi. OP9-DL1 cells are GFP⁺. (B–E) Representative Kit/CD25 flow cytometry plots (B) and absolute cell number counts of ETP (C), DN2a (D), and DN2b (E) cells within the CD45⁺GFP⁻NGFR⁺Lineage⁻CD44⁺ gate in the OP9 assay described in A. Cell counts are shown for 1,000-seeded NGFR⁺ LMPP cells; n = 3/group. (F)Zmiz1 qRT-PCR analysis of control and ZMIZ1-transduced ETPs from B compared to human ETP-ALL (LOUCY; CUTLL3) using primers that detect both mouse and human Zmiz1 mRNA; n = 3/group. (G) qRT-PCR analysis of expression of Notch1 target genes Tcf7, Gata3, and Hes1 in control and ZMIZ1-transduced ETPs from B; n = 3/group. (H) Experimental strategy to show in vivo effects of retroviral transduction of ZMIZ1 into LMPPs followed by intrathymic injection on generation of ETPs and other immature T cells from LMPPs. (I–L) Representative Kit/CD25 flow cytometry plots (I) and absolute counts of ETP (J), DN2a (K), and DN2b (L) cells on day 5 within the CD45.2⁺CD45.1⁻NGFR⁺Lineage⁻CD44⁺ gate after intrathymic injection as described in H; n = 9 (control) and 8 (ZMIZ1)/group. P values were based on two-sided t tests. *P < 0.05; **P < 0.01. ***P < 0.001.