Figure 3. FOXO3 Is Active and Required to Preserve the Immature State of Human AML Cell Lines.

(A)THP-1 and Mono-mac-6 (MM6) (both MLL-AF9⁺)and SKM-1 and NB4(both MLLAF9⁻) leukemia cell lines were fractionated into nuclear (N) and cytoplasmic

(C) extracts and subjected to western blotting with FOXO3, ORC2 (nuclear), and Tubulin (cytoplasmic) antibodies.

(B) MOLM-14, MM6, and SKM-1 cells were stably transduced with recombinant lentiviruses expressing either nontargeting (NT) or FOXO3 (F3-1 or F3-2) shRNAs. Cells were then subjected to western blotting with FOXO3 and Tubulin antibodies or (C) counted either everyday (SKM-1 and NB4) or every 2 days (MOLM-14 and MM6) following stable expression of designated shRNAs (day 6 MOLM-14, NT versus F3-1, *p = 0.0003; NT versus F3-2, **p < 0.0001; day 6 MM6, NT versus F3-1, *p < 0.0001; NT versus F3-2, **p = 0.0002; day 4 SKM-1, NT versus F3-1, *p = 0.0062; NT versus F3-2, **p = 0.0083; day 4 NB4, NT versus F3-1, *p = 0.0003; NT versus F3-2, **p = 0.0058). Data are represented as the mean ± SD.

(D) Transduced MOLM-14 and SKM-1 cells were analyzed by flow cytometry for human CD11b expression (MOLM-14, NT versus F3-1, *p< 0.0001; NT versus F3-2, **p < 0.0001; SKM-1, NT versus F3-1, *p < 0.0001; NT versus F3-2, **p = 0.0115). Data are represented as the mean ± SD.

(E) Flow cytometric analysis of transduced MM6 cells incubated with pHrodo particles.

(F) May-Grünwald Giemsa staining of transduced MOLM-14, SKM-1, and NB4 cells.

(G) Flow cytometric analysis of transduced SKM-1 cells stained with Annexin V and CD11b (*NT shRNA versus FOXO3 shRNA-1, CD11b⁺, p < 0.0001; **NT shRNA versus FOXO3 shRNA-2, CD11b⁺, p = 0.0007; n = 3). Data are represented as the mean ± SD.

NT = NT shRNA, F3-1 = FOXO3 shRNA-1, and F3-2 = FOXO3 shRNA-2; n = 3. See also Figure S3.