Fig. 3: Tal1 and Lmo2 promote DN3 label retention during leukemogenesis.

A Design of in vivo label retention studies using the doxycycline-inducible H2B-GFP mouse reporter. B Representative flow cytometry plots of WT (top row) and preleukemic Tal1/Lmo2 (bottom row) thymi after pulse-chase. Left column, gated on single cells, middle column gated on DN, right column gated on DN3 cells. Quantification of (C), percentage of DN3 cells scored as GFP^HI after the 2-week chase period (D), total DN3 GFP^HI thymocytes and (E), total GFP^LO DN3 thymocytes in WT (n = 17; gray), preleukemic Tal1 (n = 11; green), preleukemic Lmo2 (n = 16; green), preleukemic Tal1/Lmo2 (n = 12; green) mice, or leukemic mice chased for 2–3 weeks (T-ALL; n = 8; red). F Representative FACS plots of T-ALL cells after pulse-chase showing co-staining for DN3a/DN3b subpopulations. From left to right, cells were gated on singlets, DN, total DN3 cells, DN3 GFP^HI and DN3 GFP^LO. First DN3 plot gated shows label retention of all DN3 cells and second plot shows DN3a/b distribution among DN3 cells. Gating on DN3 GFP^HI and DN3 GFP^LO shows that dormant cells are significantly enriched among the DN3a subpopulation. G Left panel, quantification of the percentage of total, GFP^HI or GFP^LO leukemic DN3 cells that scored as DN3a or DN3b (n = 5). Right panel, pie chart showing the relative frequencies of each leukemic subpopulation, with GFP^HI cells comprising a minor fraction (0.64%) of the DN3a subpopulation. All error bars represent mean \(\pm \,\)SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.