Figure 5.
The cytotoxicity of AS1842856 in B-ALL depends on FOXO1 protein but extends beyond its transcriptional inhibition. (A) Treatment with dTAG-13 but not AS1842856 induces degradation of FOXO1-dTAG FP. FOXO1-dTAG clones were treated with dTAG-13 and/or AS1842856, at concentrations of 500 nM and 100 nM, respectively; 24 hours after treatment, the cells were harvested and FOXO1/FOXO1-dTAG expression was measured by immunoblot using anti-FOXO1 antibody. (B) Apoptosis induction in 018Z WT and FOXO1-dTAG clones was measured by propidium iodide (PI)/annexin V-allophycocyanin (APC) staining and subsequent flow cytometry 3 days after treatment with dTAG-13 at a concentration of 500 nM, expressed as percentage of PI+, annexin V–positive, and PI+/annexin V–positive cells (n = 3). Specific apoptosis was calculated by normalization to respective DMSO controls. Statistical significance was analyzed by ratio paired t test calculating the difference between treatment with AS1842856 and AS1842856 + dTAG-13 (WT, P = .402; FOXO1-dTAG clones, P = .005; n = 3). (C-D) Effect of FOXO1 degradation on the sensitivity of B-ALL cells to AS1842856. The cytotoxic effect was measured by a 6-day MTT test. To this end, 018Z FOXO1-dTAG cells were incubated in complete medium and treated with 500 nM of dTAG-13 or vehicle (DMSO). Two hours later, AS1842856 was added at gradually decreasing concentrations with a dilution factor of 2. The dose-effect data were fitted by nonlinear regression model. The statistical significance of the IC50 values differences was calculated by Student t test (D). (E-H) 018Z-FOXO1-dTAG cells were treated for 24 hours with 30 nM of AS1842856 (+) or vehicle DMSO (−). FOXO1 degradation was initiated by treatment with 500 nM of dTAG-13. (E) Expression of GSK3A, GSK3B, CTNNB1, FOXO1-dTAG FP, and TUBB was measured by immunoblot. The most representative of 3 independent experiments is shown. (F-H) Expression of GSK3A, GSK3B, and CTNNB1 was quantified by ImageJ software. To calculate the relative protein expression intensities of immunoblot signals (n = 3), results were normalized to TUBB. The statistical significance was analyzed by 2-sided, nonpaired t test.

The cytotoxicity of AS1842856 in B-ALL depends on FOXO1 protein but extends beyond its transcriptional inhibition. (A) Treatment with dTAG-13 but not AS1842856 induces degradation of FOXO1-dTAG FP. FOXO1-dTAG clones were treated with dTAG-13 and/or AS1842856, at concentrations of 500 nM and 100 nM, respectively; 24 hours after treatment, the cells were harvested and FOXO1/FOXO1-dTAG expression was measured by immunoblot using anti-FOXO1 antibody. (B) Apoptosis induction in 018Z WT and FOXO1-dTAG clones was measured by propidium iodide (PI)/annexin V-allophycocyanin (APC) staining and subsequent flow cytometry 3 days after treatment with dTAG-13 at a concentration of 500 nM, expressed as percentage of PI+, annexin V–positive, and PI+/annexin V–positive cells (n = 3). Specific apoptosis was calculated by normalization to respective DMSO controls. Statistical significance was analyzed by ratio paired t test calculating the difference between treatment with AS1842856 and AS1842856 + dTAG-13 (WT, P = .402; FOXO1-dTAG clones, P = .005; n = 3). (C-D) Effect of FOXO1 degradation on the sensitivity of B-ALL cells to AS1842856. The cytotoxic effect was measured by a 6-day MTT test. To this end, 018Z FOXO1-dTAG cells were incubated in complete medium and treated with 500 nM of dTAG-13 or vehicle (DMSO). Two hours later, AS1842856 was added at gradually decreasing concentrations with a dilution factor of 2. The dose-effect data were fitted by nonlinear regression model. The statistical significance of the IC50 values differences was calculated by Student t test (D). (E-H) 018Z-FOXO1-dTAG cells were treated for 24 hours with 30 nM of AS1842856 (+) or vehicle DMSO (−). FOXO1 degradation was initiated by treatment with 500 nM of dTAG-13. (E) Expression of GSK3A, GSK3B, CTNNB1, FOXO1-dTAG FP, and TUBB was measured by immunoblot. The most representative of 3 independent experiments is shown. (F-H) Expression of GSK3A, GSK3B, and CTNNB1 was quantified by ImageJ software. To calculate the relative protein expression intensities of immunoblot signals (n = 3), results were normalized to TUBB. The statistical significance was analyzed by 2-sided, nonpaired t test.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal