Anti cd117 microbeads

Retroviral transduction of c-Kit-enriched bone marrow (anti-CD117 microbeads; Miltenyi Biotec, Auburn, CA, USA) with NICD in the MSCV-IRES-GFP (MIG) retrovirus (8 (link)) was performed as previously described (17 (link)). Recipient mice were transplanted by retro-orbital injection following a split dose of 11.0 Gy of irradiation. For T-ALL rescue, Nr4a1 and Dnmt3a were cloned into MSCV-IRES-mCherry (MIC) to transduce primary GFP+ T-ALL cells. 4×10⁴ GFP+mCherry+ cells were transplanted into secondary recipients. For secondary transplantations, leukemic cells were transplanted into sublethally irradiated (6.0 Gy) mice. The TtRMPVIR retroviral backbone (Addgene, Cambridge, MA, USA) was modified to place Nr4a1-2A-mCherry under the control of a tetracycline-responsive promoter. Sample size was calculated based on published studies for NICD transduction and transplantation (8 (link), 18 (link)) to provide at least 80% power to compare a median survival difference of 25% based on two-sided two-sample test for proportions (p<0.05). NICD transduction and transplantation was performed independently for primary mice five times.

Single-cell suspensions were derived from mechanical disruption of mouse bone marrow, spleen, thymus and fetal liver in PBS supplemented with 2% fetal calf serum (FCS, Sigma). For FACS analysis of spleen, fetal liver and peripheral blood, red blood cells were lysed with ACK buffer. Nonspecific antibody binding was blocked by incubation with 20 µg/ml Rat IgG (Sigma) for 15 min. Cells were incubated with primary antibodies for 45 min and streptavidin conjugates, when applicable, for 15 min on ice. The antibodies used in this study are listed in Supplementary Table 1. Lineage-negative cells were defined by lack of expression of Gr-1, TER-119, CD4, CD8, B220 and CD11b (except for fetal liver samples). For intracellular Ki67 and DAPI staining in HSC, following staining of surface antigens, bone marrow cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% saponin prior to addition of anti-Ki67 (BD Biosciences) and 2 µg/ml DAPI. For sorting of rare HSC populations, bone marrow was enriched for stem and progenitor cells via magnetic selection with anti-CD117 microbeads (Miltenyi Biotec) prior to antibody staining and sorting. Stained cells were quantified using a BD Fortessa analyzer or isolated with a BD ARIA II. FlowJo software (Treestar) was used to generate flow cytometry plots, histograms and calculate mean fluorescence intensities.

Seven- to twelve-week-old female or male CD45.2⁺Cd19-Cre^+/−Dnmt3a^fl/fl were used as donors. Femurs, tibias, hips, and spines were isolated from donor mice, crushed, and ACK-lysed. Cells were enriched with anti-CD117 microbeads (Miltenyi Biotec, Bergish Gladbach, Germany) and then sorted to collect LSK (lineage^– Sca-1⁺ c-Kit⁺) cells a panel of antibodies as described in Supplementary Materials and Methods.