RUNX/CTCF represses CD25 ⁺ cell generation in LPs, whereas RUNX/Mediator promotes DN2 development after Notch stimulation. (A) Schematic of the stage-specific deletion of Cbfb, Ctcf, or Med12 in LP and Phase 1 cells using the CRISPR/Cas9 system. (B) Retroviral vectors encoding sgRNAs were introduced into Cas9-LPs. 5 days after sgRNA introduction, hNGFR⁺CD45⁺ sgRNA-transduced cells were gated and analyzed for CD44 and CD25 expression. (C and D) Percentage (C) and relative number (D) of CD25⁺ cells among hNGFR⁺CD45⁺ sgRNA-transduced cells from B are shown with SD. (E) 1 day after sgRNA introduction, LPs were transferred onto OP9-DLL4 stromal cells and cocultured for 2 days. hNGFR⁺CD45⁺ sgRNA-transduced cells were gated and analyzed for CD44 and CD25 expression. (F and G) Percentage (F) and relative number (G) of CD25⁺ cells among hNGFR⁺CD45⁺ sgRNA-transduced cells from E are shown with SD. Data in B and E are representative of three independent experiments. Data in C, D, F, and G represent mean values from three independent biological replicates. The data were analyzed by one-way ANOVA with Dunnett’s multiple comparisons (C, D, F, and G). For LP (Notch-); C, **adjusted P < 0.0001 for sgCbfb; **adjusted P = 0.0064 for sgCTCF. For LP (Notch-); D, **adjusted P < 0.0001 for sgCbfb; **adjusted P = 0.0064 for sgCTCF. For Phase 1 (Notch+); F, **adjusted P = 0.0046 for sgCbfb; **adjusted P = 0.0002 for sgCTCF; **adjusted P = 0.0362 for sgMed12. For Phase 1 (Notch+); G, **adjusted P = 0.0049 for sgCbfb; **adjusted P = 0.0002 for sgCTCF; **adjusted P = 0.0360 for sgMed12. SD, standard deviation.