AE/N-Ras^G12D cells have enhanced self-renewal and improved in vivo engraftment partly as a result of Bcl-2 up-regulation. (A) Top: CD34⁺ umbilical cord blood cells were retrovirally transduced with vector control (CON) or AE and expanded for 2 weeks. Cells were then collected for blotting of indicated proteins. Bottom: Protein lysates from AE and AE/N-Ras^G12D cells were blotted for the indicated proteins. (B) Cells were analyzed by flow cytometry for expression of annexin V and uptake of 7-amino-actinomycin D (7-AAD). (C) Colony formation assay was performed as described in Figure 1F with indicated cell numbers. (D) Long-term culture-initiating cells assay comparing AE/pBabe and AE/Bcl-2 cells. (E) Cells were treated with ABT-737 compound at indicated concentrations for 72 hours in triplicate, followed by WST-1 cell proliferation assay. Optical density (OD) values obtained from each experiment were normalized to vehicle control (0μM group). Averages from multiple experiments are shown: n = 5 in parent AE and AE/N-Ras cytokine(−); n = 3 in AE/N-Ras cytokine(+). Bars represent SD. *P < .05. (F) AE cells were transduced with viruses containing an empty vector or the N-Ras^G12D gene, followed by injection of 5 × 10⁵ cells into the left femur of NSG mice. Initial transduction was approximately 20% in both groups. Six weeks after injection, bone marrow aspiration was performed in the injected bone to determine engraftment; n = 6 in each group. (●) represents one mouse. White bars represent average. (G-H) AE/N-Ras^G12D cells were injected intrafemorally into NSS mice (G) or NSG mice (H). Total bone marrow cells from injected and noninjected bones as well as total spleen cells were obtained by crushing the organs, followed by immunostaining with antibodies against indicated surface markers and flow cytometric analysis.

AE/N-Ras^G12D cells exhibit various features of oncogenic transformation. (A) The “cytokine depletion protocol” for establishing AE/N-Ras^G12D cells. (B) Western blotting for the indicated proteins. (C) AE and AE/N-Ras^G12D cells were plated at 0.5 × 10⁶/mL. Cells were counted twice a week, followed by replating at the initial density. (D) AE/N-Ras^G12D cells grown with cytokine supplementation were plated at 0.5 × 10⁶/mL. AE/N-Ras^G12D cells grown in the absence of cytokines were plated at 0.5 × 10⁶/mL or 1 × 10⁶/mL. Cells were counted twice a week, followed by replating at the initial density. (E) AE and AE/N-Ras^G12D cells were stained with allophycocyanin-conjugated anti-CD34 antibody and analyzed by flow cytometry. (F) One representative experiment showing enhanced colony-forming and replating abilities of AE/N-Ras^G12D cells. CD34⁺ AE and AE/N-Ras^G12D cells were plated in methylcellulose media at 5 × 10⁴ cells/mL per plate in triplicate. The number of colonies was scored after 14 days. Cells were collected, washed with phosphate-buffered saline, and replated at 5 × 10⁴ cells/mL per plate in triplicate. (G) Cells collected from methylcellulose media were immunostained with antibodies against the indicated surface markers, followed by flow cytometric analysis. (H) A total of 1 × 10⁵ cells were cytocentrifuged onto glass slides and Wright-Giemsa stained. Pictures were photographed using Leica LAS software with LeicaDMI6000B Inverted Research Microscpoe, 40× dry objective and Leica DFC290HD camera. Bars represent 10 μm.