FZR1 knockdown sensitizes human B-ALL cells to DNA damage–induced cell death and mitotic catastrophe. (A) Immunoblot of FZR1 in NALM-6 and RS4;11 cells transduced with lentivirus expressing tetracycline-inducible control (C) shRNA or shRNA specific to FZR1 (shFZR1_1 and shFZR1_2) after 72-hour and 120-hour treatment with 1 μg/mL tetracycline. (B) Time-course cell proliferation assay of NALM-6 and RS4;11 cells with tetracycline-induced shC or shFZR1 treated with tetracycline (0.25 μg/mL for NALM-6 cells and 0.10 μg/mL for RS4;11). Trypan blue–negative cells were counted at described time points. P values were calculated by comparing with untreated controls. (C) Apoptosis analysis of NALM-6 and RS4;11 cells with tetracycline-induced shC or shFZR1 after treatment with tetracycline and doxorubicin. Cells were treated with tetracycline (0.125 μg/mL for NALM-6 and 0.10 μg/mL for RS4;11) for 5 days and then treated with doxorubicin (0.125 μM for NALM-6 and 0.25 μM for RS4;11) for 36 hours. Annexin V–positive cells were counted by flow cytometry. (D) Costaining of γH2A.X and cleaved caspase-3 in NALM-6 cells with tetracycline-inducible shC or shFZR1 (shFZR1_1). Cells were treated with 0.125 μg/mL tetracycline for 5 days and then treated with 0.5 μM doxorubicin. (E) Flow cytometric analysis of 5-ethynyl-2′-deoxyuridine (EdU) incorporation combined with DNA content analysis with propidium iodide (PI) staining in NALM-6 cells with tetracycline-inducible shC or shFZR1 (shFZR1_1). Cells were treated with 0.125 μg/mL tetracycline for 5 days and then treated with 0.125 μM doxorubicin. Graphs in panels D and E are representative examples from 3 experiments. Except for panel A, results are expressed as mean ± SD (n = 3). *P < .05, ** P < .01, ***P < .001, ****P < .0001.