Figure 4. Primary AMLs Derived from Patients Separate into Distinct Clusters of FOXO Activity.

(A) BM cells derived from patients with AML were stained with human lineage cocktail and human CD34 (both BD Biosciences), and then lineage^low, CD34⁺ cells were isolated by flow cytometry. Nuclear (N) and cytoplasmic (C) extracts of total MNBCs (TBM) and lineage^low, CD34⁺ cells were subjected to western blotting with FOXO3, ORC2, and Tubulin antibodies.

(B) Lineage^low, CD34⁺ cells from three AML patients analyzed as described above.

(C) Patient samples #1 and #6 were transduced with recombinant lentiviruses expressing either NT shRNA or FOXO3 shRNA-1, then grown in liquid culture for 8 days, and then assessed for CD11b expression. Bar graphs are represented as the mean ± SD.

(D) Transduced patient samples #1 and #6 cells were placed in methylcellulose supplemented with human cytokines. Graph represents the enumeration of colonies formed after 8 days of culture. Data are represented as the mean ± SD.

(E) Transduced cells from patient sample #1 stained with Wright-Giemsa after 8 days of liquid culture.

(F) Gene list comprising the FOXO-specific gene signature generated from comparing the gene expression array data of murine lineage^low, Sca-1⁺, cKit⁺ (LSK) cells in animals without (+/+) and with (Δ/Δ) FoxO1/3/4 deletion (comprehensive gene set located in Table S1).

(G) Hierarchical cluster analysis based on the overlap of the murine FOXO gene signature stratified over the gene expression array data of 436 individual primary AML samples.

See also Figure S4 and Table S1.