Extended Data Fig. 2: Loss of TCF-1′s L1 domain limits DN1 to DN2 transition.

a) Thy1⁺ CD25⁺ cells (b) DN1 (CD44⁺ CD25⁻), DN2 (CD44⁺CD25⁺), and DN3 (CD44- CD25+) in OP9-DLL1 co-cultures of Tcf7 cKOs transduced with EV, WT, or mutant TCF-1 (ΔL1-7) on day 13. c) Quantification of Thy1⁺ CD25⁺ cells (top) and DN2s, and DN3s by CD44 and CD25 surface expression (bottom) in OP9-DLL1 co-cultures of Tcf7 cKOs transduced with EV, WT, or mutant TCF-1 on day 13. d) DN1 (CD44⁻ CD25⁻), DN2 (CD44⁺ CD25⁺), and DN3 (CD44⁻ CD25⁺) cells in co-cultures of wild type (WT) ckit⁺ bone marrow (BM) progenitors transduced with WT TCF-1, EV, or mutant TCF-1 (ΔL1-L7) (GFP⁺) on OP9-DLL1 cells at day 5. e) Frequency of GFP⁺ (transduced) and GFP⁻ (un-transduced) DN2s and DN3s in (D) (top). Analysis of ratio of GFP+ to GFP- Thy1⁺ CD25⁺ cells in (d) (middle). Frequency of GFP+ and GFP⁻ Thy1⁺ CD25⁺ cells in (d) (bottom). f) DN1 (CD44⁻ CD25⁻), DN2 (CD44⁺ CD25⁺) and DN3 (CD44⁻ CD25⁺) cells in WT ckit⁺ BM progenitors transduced with WT TCF-1, ΔL1, or EV (GFP⁺) on OP9-DLL4 after 5 days. g) DN1 (CD44⁺ CD25⁻), DN2 (CD44⁺ CD25⁺), and DN3 (CD44⁻ CD25⁺) cells in WT ckit⁺ BM progenitors transduced with WT TCF-1 (GFP⁺) on OP9-DLL1 cells (left) and OP9 cells (right) for 5 days. h) Histogram depicting TCF-1 intracellular flow cytometry in Tcf7 cKO progenitors un-transduced (Vex⁻) or transduced with WT TCF-1, ΔL1, or EV (Vex⁺) as well as WT TCF-1 sufficient progenitors. i) Frequency of B220⁺ Vex⁺ (transduced) cells in Tcf7 cKO OP9-DLL1 at day 5 (left) and 13 (right). j) Number (left) and frequency (right) of CD11b⁺ CD25⁻ cells at day 7 in Tcf7 cKO OP9-DLL1 co-cultures. All cells (a-j) are pre-gated on SSC-A/FSC-A, Singlets, Live cell (Viability-), CD45+ and for a,b,c,i,j Vex+ and all data are representative of at least 3 independent experiments. In c, e, i,j, bars show mean from n = 2 independent animals, dots represent individual data points. In c, i, P values are determined by one-way ANOVA followed by Dunnett’s multiple comparison test with WT TCF-1 (P45) as a control. *P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.001.