Characterization of the effects of genetic depletion and pharmacological inhibition of FOXO1 in a BCR::ABL1⁺ B-ALL mouse model. (A-B) AS1842856 downregulates Myc and Ccnd3 messenger RNA expression and induces cell cycle perturbations in BCR::ABL1–transformed murine pre–B cells. (A) The cells were treated with 70 nM AS1842856. The cell cycle distribution was measured by propidium iodide staining after 24 and 48 hours (n = 3). The data are shown as mean ± standard deviation. (B) Myc and Ccnd3 expression was measured by quantitative reverse transcription PCR (n = 3) and calculated using the 2^–△△Ct method. The housekeeping gene Rpl13a was used for normalization. (C-D) BCR::ABL1⁺ CreERT2/Foxo1^fl/fl murine B-ALL cells were treated with 200 nM 4-OHT. Protein expression of FOXO1, MYC, CCND3, and Cl. Casp3 was monitored by immunoblot. A representative of 2 independent experiments is shown. (D) Quantification of immunoblots. Protein expression of FOXO1, CCND3, and MYC was divided by the corresponding beta-actin (ACTB) levels and normalized to the protein of interest-to-ACTB ratio at day 0 (100%; n = 2). (E) BCR::ABL1⁺ Cre-ERT2 Foxo1^fl/fl murine B-ALL cells were treated with 200 nM 4-OHT or equivalent amounts of EtOH vehicle. Cells were stained for Il7ra surface expression or isotype control and analyzed by flow cytometry at the indicated time points. Data shown are representative of 3 independent experiments. (F) BCR::ABL1⁺ Cre-ERT2 Foxo1^fl/fl murine B-ALL cells were treated with 70 nM AS1842856 or equivalent amounts of DMSO vehicle. Cells were stained for Il7ra surface expression or isotype control and analyzed by flow cytometry at the indicated time points. Data shown are representative of n = 3. Cl. Casp3, cleaved caspase-3; EtOH, ethyl alcohol; Iso, isotype; Rel, relative.