Nod scid il2rg mice

All animal studies were performed in accordance with UCSD and NIH-equivalent ethical guidelines and were approved by the Institutional Animal Care and Use Committee. Three AML primagraft models were established from AML LSC-enriched cell fractions (1–2 × 10⁵ CD34⁺ cells) transplanted intrahepatically into neonatal Rag2^−/−γ_c^−/− as previously described (Abrahamsson et al., 2009 (link)), or intravenously into sublethally irradiated adult (6–8 weeks old) NOD/SCID-IL2RG mice (NSGS, Jackson Laboratory) (Figure S6). AML-engrafted mice were dosed intravenously with 17S-FD-895 (5–10 mg/kg) or vehicle (15–20% DMSO in PBS) three times over a two-week period (day 1, day 7, and day 14). After treatment, hematopoietic tissues were analyzed as described in Supplemental Experimental Procedures.
Additional study design, methods, and detailed statistical analyses are described in Supplemental Experimental Procedures.

In vivo experiments were done in accordance with the HMRI institutional guidelines for the use of laboratory animals. To determine tumorigenicity of regenerated SP cells, 8- to 10-week-old NOD/SCID IL2rg−/− mice (Jackson Laboratory, Bar Harbor, ME, USA) (six mice per group) were injected subcutaneously with 0.1 × 10⁶ SP or NSP cell populations isolated from the RPMI8226 GL cell line and suspended in Matrigel (BD Biosciences). Tumor engraftment was monitored weekly by whole-body bioluminescence imaging. Luciferin substrate (Gold Bio Technology, St. Louis, MO, USA) was injected intraperitoneally (IP) and whole-body imaging performed on the Xenogen IVIS system (PerkinElmer, Waltham, MA, USA), as previously reported.^{20 (link)} Mice were euthanized on day 35 in accordance with institutional guidelines. Bioluminescence signal intensity representative of tumor sizes was quantified by Living Image 3.1 (PerkinElmer).
To determine the ability of SP and NSP cells to infiltrate the mouse bone marrow (BM), mice (same as above) were injected via the lateral tail vein with 0.2 × 10⁶ sorted SP or NSP cells. Tumor engraftment was monitored using bioluminescence for 3 weeks. Mice were then euthanized and bone marrow cells were flushed with PBS from femora and tibiae using a 25-gauge needle. The percentage of GFP-positive cells was determined by flow cytometry.

A piece of HCC was obtained from a patient at the Keck Medical Center of University of Southern California with informed consent. Tumor collection and use were approved by Institutional Review Board of the University of Southern California (HS-16-00392). Freshly excised HCC was cut into 8 mm³ pieces and transplanted subcutaneously in the right flank of 6-wk old male NSG^TM mice (NOD-SCID-Il2rg−/− mice, Jackson Laboratory). On the third and fourth passages, the mice were grouped randomly and treated intraperitoneally with vehicle, BC (20 mg/kg), CCI-779 (5 mg/kg) or both compounds. The compounds were solubilized by a four times concentrated solution containing 50% cremophor EL (EMD Millipore) and 50% ethanol and kept frozen at −20°C. Prior to treatment, the compounds were thawed and diluted four times with bacteriostatic saline. Tumor volumes were measured with a caliper at indicated days and calculated as (LXW²)/2.

NOD/SCID/IL2rg−/−SGM3 (NSGS) mice were generated by crossing NOD/SCID/IL2rg−/− mice (Jackson Laboratory, Bar Harbor, Maine) with NOD/SCID mice with transgenic expression of hSCF, hGM-CSF and hIL-3 (a kind gift from Dr. C. Eaves), and were used as recipients^{34 (link)}. Human CB CD34+ cells were transduced with Vector, wild-type or mutant CBL (coexpressing GFP) together with AML1-ETO (coexpressing Thy1.1), and were injected by intrafemoral injection into sublethally irradiated (250cGy from a cesium source) 6- to 8-week-old male or female mice. The recipient mice were euthanized 12 – 16 weeks after transplantation for analyses. We used human cord blood cells derived from a single donor for each experiment, and performed three independent experiments using 6 recipient mice in total for each group (three mice for cord blood 1, one mouse for cord blood 2, and two mice for cord blood 3). Randomization and blinding were not performed in this study.