Figure 2.
Figure 2. FOXO1 knockdown induces growth arrest and apoptosis in BCP-ALL. (A) Efficient shRNA-mediated knockdown of FOXO1. BCP-ALL and cHL (L428, U-HO1) cell lines were transduced with lentiviral vectors expressing scrambled (Scr), FOXO1-targeting shRNA-81 (shF1-81) or shRNA targeting FOXO1, FOXO3A, and FOXO6 (shF1-136). Transduced cells were selected by incubation with puromycin, and FOXO1 expression was assessed by immunoblot. A representative of 2 independent experiments is shown. (B) Antileukemia effect of FOXO1 knockdown. The dynamic of the RFP+ population was measured every 3 days starting at day 3 after transduction. The proportion of RFP+ cells on first measurement (day 3) was taken as 100%. Data represent mean ± SD of 3 independent transductions. (C-E) Cells transduced with Scr or shRF1-81 vectors were sorted on day 4 after transduction. On day 5 cell cycle phase distribution (C) and cell death (D) were measured by PI and by annexin V–fluorescein isothiocyanate/PI staining, respectively. Mean percentages ± SD, 2 independent experiments, each measured twice (C). Specific apoptosis was calculated as SA(%) = 100 × (AE − AC)/(100 − AC), where AE equals the percentage of apoptotic cells in the experimental group and AC equals the percentage of apoptotic cells in the control group (N = 3) (D). (E) Induction of caspase-3 cleavage by FOXO1 depletion. Cleaved CASP3 was detected by immunoblot in lysates of sorted BCP-ALL cell lines. A representative image of 2 independent experiments is shown. (F-G) Overexpression of wild-type FOXO1 harboring wobbled shRNA-targeted sequence (wtF1wb) protects 018Z and NALM-6 cells from the cytotoxic effect of shF1-81. Cells were transduced with SF-LV-cDNA-eGFP empty vector (EV) or with SF-LV-wtF1wb-expressing vector. In 4 to 6 days, the cells were transduced with shF1-81 or Scr vectors expressing the fluorescent marker RFP. For FOXO1 protein analysis, cells were sorted by fluorescence-activated cell sorting (FACS) on day 3 after the second transduction (F). The percentage of the GFP+/RFP+ population was measured at indicated time points after second transduction. Data are shown as mean ± SD of 2 independent experiments (G). *P < .05; **P < .01; ***P < .001; ****P < .0001.

FOXO1 knockdown induces growth arrest and apoptosis in BCP-ALL. (A) Efficient shRNA-mediated knockdown of FOXO1. BCP-ALL and cHL (L428, U-HO1) cell lines were transduced with lentiviral vectors expressing scrambled (Scr), FOXO1-targeting shRNA-81 (shF1-81) or shRNA targeting FOXO1, FOXO3A, and FOXO6 (shF1-136). Transduced cells were selected by incubation with puromycin, and FOXO1 expression was assessed by immunoblot. A representative of 2 independent experiments is shown. (B) Antileukemia effect of FOXO1 knockdown. The dynamic of the RFP+ population was measured every 3 days starting at day 3 after transduction. The proportion of RFP+ cells on first measurement (day 3) was taken as 100%. Data represent mean ± SD of 3 independent transductions. (C-E) Cells transduced with Scr or shRF1-81 vectors were sorted on day 4 after transduction. On day 5 cell cycle phase distribution (C) and cell death (D) were measured by PI and by annexin V–fluorescein isothiocyanate/PI staining, respectively. Mean percentages ± SD, 2 independent experiments, each measured twice (C). Specific apoptosis was calculated as SA(%) = 100 × (AE − AC)/(100 − AC), where AE equals the percentage of apoptotic cells in the experimental group and AC equals the percentage of apoptotic cells in the control group (N = 3) (D). (E) Induction of caspase-3 cleavage by FOXO1 depletion. Cleaved CASP3 was detected by immunoblot in lysates of sorted BCP-ALL cell lines. A representative image of 2 independent experiments is shown. (F-G) Overexpression of wild-type FOXO1 harboring wobbled shRNA-targeted sequence (wtF1wb) protects 018Z and NALM-6 cells from the cytotoxic effect of shF1-81. Cells were transduced with SF-LV-cDNA-eGFP empty vector (EV) or with SF-LV-wtF1wb-expressing vector. In 4 to 6 days, the cells were transduced with shF1-81 or Scr vectors expressing the fluorescent marker RFP. For FOXO1 protein analysis, cells were sorted by fluorescence-activated cell sorting (FACS) on day 3 after the second transduction (F). The percentage of the GFP+/RFP+ population was measured at indicated time points after second transduction. Data are shown as mean ± SD of 2 independent experiments (G). *P < .05; **P < .01; ***P < .001; ****P < .0001.

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