In vitro transformation of primary bone marrow cells by BCR-ABL in Skp2^+/+ versus Skp2^−/− bone marrow. (A) Flow cytometric analysis of non–5-FU-treated Skp2^+/+ and Skp2^−/− mice bone marrow cells for stem/multipotent progenitor population (left) and lineage surface markers (right). Significant differences (P < .05) are indicated by an asterisk. (B) Immunoblot showing Skp2 expression from bone marrow cells of Skp2^+/+, Skp2^+/−, and Skp2^−/− littermates. Actin was used as a loading control. (C) Comparison of B-cell transformation by BCR-ABL between Skp2^+/+ and Skp2^−/− bone marrow cells. C57/BL6 bone marrow cells were transduced with p210^BCR-ABL or empty vector retroviral supernatant and the indicated numbers of transduced viable cells were plated in Whitlock-Witte culture medium in triplicate on stromal cells (10⁶ cells/well) derived from untransduced marrow. Wells were scored as positive when the numbers of viable nonadherent cells reached 10⁶/well. No growth was observed in Skp2^+/+ or Skp2^−/− bone marrow cells transduced with empty vector retrovirus. Data shown here are representative of 3 independent experiments. (D) Comparison of myeloid colony formation between Skp2^+/+ and Skp2^−/− bone marrow cells. Bone marrow cells were transduced with p210^BCR-ABL or empty vector retrovirus and plated in methylcellulose in the presence or absence of cytokines. The histogram shows the average percentage of remaining colonies for Skp2^−/− marrow cells (considering Skp2^+/+ as 100%) from triplicate assays from 3 independent experiments. The difference between Skp2^+/+ and Skp2^−/− mice is significant (P < .001). No colonies were recovered from cells transduced with empty vector and grown in the absence of cytokines. Mice used for in vitro assays are not treated with 5-FU. Error bars represent SE.