Mnt deletion in vitro promotes the death of MLL::AF9 AML CLs. (A) Protocol. Fetal liver cells containing hemopoietic stem and progenitor cells (HSPCs) from E14 Mntfl/fl, CreERT2 or Mntfl/flCreERT2 embryos (C57BL/6-Ly5.2) were infected with either MLL::AF9/GFP or control GFP MSCV virus and transplanted into sublethally irradiated (7.5 Gy) C57BL/6-Ly5.1 recipients (2 × 106 cells per mouse). Mice that developed leukemia were autopsied, subjected to hemopoietic analysis, and their bone marrow (BM) and spleen cells were cryopreserved. (B) Robust expression of MNT and MYC proteins in BM of exemplar primary (T0) MLL::AF9 AML-bearing mice (identified by number), determined by western blot analysis. AH15 is a control Eμ-Myc lymphoma CL in which Mnt was deleted by CRISPR/Cas9 (C. J. Vandenberg and S. Cory, unpublished data, November 2014). Molecular weight markers (kilodalton) are indicated. The results are representative of independent experiments. (C) Polymerase chain reaction analysis showing the deletion of floxed Mnt alleles by CRE-ERT2 recombinase after treatment with 0.5 μM 4-OHT, indicated by the appearance of a 386 bp MntΔ fragment and the concomitant loss of a 579 bp Mntfl/fl fragment. The results are shown for 2 representative Mntfl/flCreERT2/MLL::AF9 AML CLs (2205 and 2207; pink) and 2 control (nondeletable) CreERT2/MLL::AF9 AML CLs (2210 and 2244; blue). The DNA ladder (base pair) is indicated. (D) Flow cytometric analysis of cell viability following treatment with 4-OHT or vehicle (ethanol; –4-OHT) for 24 hours. Representative plots are shown for the indicated Mntfl/flCreERT2/MLL::AF9 and control CreERT2/MLL::AF9 AML CLs. The cells were stained with PI; viable cells are PI-negative. (E) Mnt deletion provoked death of the MLL::AF9 AML cells. Multiple independent Mntfl/flCreERT2/MLL::AF9 (pink) and control CreERT2/MLL::AF9 (blue) AML CLs were treated in duplicate for 24 hours with 0.5 μM 4-OHT, then washed and resuspended in normal medium and incubated for a further 24 hours before determining the percentage of PI-negative cells by flow cytometry. Two experiments were performed, and a total of 7 Mntfl/flCreERT2/MLL::AF9 and 7 CreERT2/MLL::AF9 AMLs were tested (see legend); 2 of the former and 3 of the latter were tested in both experiments. The results are presented as mean ± standard error of the mean (SEM) relative to samples incubated without 4-OHT. Significance was determined by 1-way analysis of variance (ANOVA) with Tukey multiple comparison test (∗P ≤ .05). (F) MNT loss does not alter the expression of the MLL::AF9 fusion protein. A representative western blot analysis is shown for the CreERT2/MLL::AF9 AML and Mntfl/flCreERT2/MLL::AF9 AML CLs treated with 0.5 μM 4-OHT or vehicle (ethanol) for 48 hours with addition of a pan-caspase inhibitor (QVD-OPH; 25 μM). The expression levels of the MNT, MLL-1, and MLL::AF9 proteins are shown with actin as the loading control. FSC, forward light scatter; KO, knockout; PI, propidium iodide; WT, wild type.
Figure 1.

Mnt deletion in vitro promotes the death of MLL::AF9 AML CLs. (A) Protocol. Fetal liver cells containing hemopoietic stem and progenitor cells (HSPCs) from E14 Mntfl/fl, CreERT2 or Mntfl/flCreERT2 embryos (C57BL/6-Ly5.2) were infected with either MLL::AF9/GFP or control GFP MSCV virus and transplanted into sublethally irradiated (7.5 Gy) C57BL/6-Ly5.1 recipients (2 × 106 cells per mouse). Mice that developed leukemia were autopsied, subjected to hemopoietic analysis, and their bone marrow (BM) and spleen cells were cryopreserved. (B) Robust expression of MNT and MYC proteins in BM of exemplar primary (T0) MLL::AF9 AML-bearing mice (identified by number), determined by western blot analysis. AH15 is a control Eμ-Myc lymphoma CL in which Mnt was deleted by CRISPR/Cas9 (C. J. Vandenberg and S. Cory, unpublished data, November 2014). Molecular weight markers (kilodalton) are indicated. The results are representative of independent experiments. (C) Polymerase chain reaction analysis showing the deletion of floxed Mnt alleles by CRE-ERT2 recombinase after treatment with 0.5 μM 4-OHT, indicated by the appearance of a 386 bp MntΔ fragment and the concomitant loss of a 579 bp Mntfl/fl fragment. The results are shown for 2 representative Mntfl/flCreERT2/MLL::AF9 AML CLs (2205 and 2207; pink) and 2 control (nondeletable) CreERT2/MLL::AF9 AML CLs (2210 and 2244; blue). The DNA ladder (base pair) is indicated. (D) Flow cytometric analysis of cell viability following treatment with 4-OHT or vehicle (ethanol; –4-OHT) for 24 hours. Representative plots are shown for the indicated Mntfl/flCreERT2/MLL::AF9 and control CreERT2/MLL::AF9 AML CLs. The cells were stained with PI; viable cells are PI-negative. (E) Mnt deletion provoked death of the MLL::AF9 AML cells. Multiple independent Mntfl/flCreERT2/MLL::AF9 (pink) and control CreERT2/MLL::AF9 (blue) AML CLs were treated in duplicate for 24 hours with 0.5 μM 4-OHT, then washed and resuspended in normal medium and incubated for a further 24 hours before determining the percentage of PI-negative cells by flow cytometry. Two experiments were performed, and a total of 7 Mntfl/flCreERT2/MLL::AF9 and 7 CreERT2/MLL::AF9 AMLs were tested (see legend); 2 of the former and 3 of the latter were tested in both experiments. The results are presented as mean ± standard error of the mean (SEM) relative to samples incubated without 4-OHT. Significance was determined by 1-way analysis of variance (ANOVA) with Tukey multiple comparison test (∗P ≤ .05). (F) MNT loss does not alter the expression of the MLL::AF9 fusion protein. A representative western blot analysis is shown for the CreERT2/MLL::AF9 AML and Mntfl/flCreERT2/MLL::AF9 AML CLs treated with 0.5 μM 4-OHT or vehicle (ethanol) for 48 hours with addition of a pan-caspase inhibitor (QVD-OPH; 25 μM). The expression levels of the MNT, MLL-1, and MLL::AF9 proteins are shown with actin as the loading control. FSC, forward light scatter; KO, knockout; PI, propidium iodide; WT, wild type.

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