Nsg2 is highly upregulated in HSCs and leukemic tissues of Tg mice and impairs hematopoietic differentiation. (A) Scatter plot comparing the transcriptomes of control and Tg IL-7Rα⁻ LSK HSCs. Transcription levels are shown on a logarithmic scale. A linear regression curve and correlation coefficient are indicated. (B) Nsg2 mRNA in IL-7Rα⁻ LSK HSCs was measured by real-time quantitative RT-PCR, and is presented as the relative expression level in arbitrary units where the mRNA level in the control was defined as 1 unit. (C) Comparison of Nsg2 mRNA levels in IL-7Rα⁻ LSK HSCs from the control and Tg BM, and in the control normal spleen as well as leukemic spleens from Tg mice. Nsg2 mRNA was measured and shown as in panel B. Among leukemic spleens, gray and black bars indicate the leukemic tissues that were diagnosed with AML and B-ALL, respectively. *Unclassified samples. (D) Murine primary c-Kit⁺ BM cells were retrovirally transduced with pMYs-IRES-EGFP empty vector or the vector expressing Nsg2. Cells were grown in liquid culture with SCF, IL-3, IL-6, GM-CSF, TPO, and Flt3L for 5 days and assessed for the percentage of c-Kit⁺ population in EGFP^high cells. (Left) Flow cytometric profiles. (Right) The ratio of c-Kit⁺ cells (percentage of EGFP⁺ cells). (E) Cells treated as in panel D were assessed using flow cytometry for the percentage of LSK in total Lin⁻EGFP^high cells. (Left) Flow cytometric profiles. (Right) The ratio of LSK cells (percentage of EGFP⁺ cells). All results are shown as the mean ± SD of independent experiments; **P < .005. (F) c-Kit⁺ BM cells from the control and Tg littermates were retrovirally transduced with pMYs-IRES-EGFP empty vector. c-Kit⁺ BM cells from control mice were retrovirally transduced with the vector expressing Nsg2. EGFP⁺ cells were sorted and then cultured in methylcellulose media as described in “Methods.” Colonies were counted and replated at 2 × 10⁴ cells every 7 days. Error bars, SD. **P < .005. (G) Representative images of colonies from the third plating as shown in panel F. Scale bars, 200 µm. EGFP, enhanced green fluorescent protein; GM-CSF, granulocyte macrophage–colony-stimulating factor; IL, interleukin; IRES, internal ribosome entry site; RT, reverse transcription; SCF, stem cell factor; TPO, thrombopoietin.