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. 2019 Apr 25;134(1):59–73. doi: 10.1182/blood.2018893982

Figure 1.

Figure 1.

Depletion of RUNX1 by shRNA diminishes RUNX1 target gene expression and induces greater in vitro lethality in mtRUNX1-expressing than wtRUNX1 AML BPCs, as well as attenuates leukemia engraftment and significantly prolongs survival of mice bearing luciferase-expressing OCI-AML5 xenografts. (A) Relative mRNA expression of RUNX1, RUNX2, RUNX3, MPO, BCL-2, and MYC in OCI-AML5 cells transduced with non-target shRNA (sh NT) or RUNX1 shRNA for 72 hours. Expression of each mRNA was normalized to GAPDH and to sh NT. (B) Immunoblot analysis of OCI-AML5 cells transduced with sh NT or RUNX1 shRNA for 72 hours. The numbers beneath the bands represent densitometry analysis. (C) Colony growth of OCI-AML5 and OCI-AML2 cells transduced with sh NT or RUNX1 shRNA for 72 hours. ***P < .005 compared with sh NT–transduced cells. (D) Percent apoptosis in OCI-AML5 and OCI-AML2 cells transduced with sh NT or RUNX1 shRNA for 72 hours. ***P < .005 compared with sh NT-transduced cells. (E) qPCR analysis for RUNX1 expression in PD mtRUNX1 AML cells (n = 3) and wtRUNX1 AML (n = 2) transduced with sh NT or RUNX1 shRNA for 72 hours. Relative mRNA expression was normalized to GAPDH and compared with sh NT expression levels. (F) PD CD34+ mtRUNX1 (n = 3) and wtRUNX1 AML (n = 2) samples were transduced with sh NT or RUNX1 shRNA for 72 hours. The percentage of nonviable cells was determined by using flow cytometry. *P < .05, ***P < .005 relative to sh NT cells. (G) Immunoblot analysis after 72 hours of 100 ng/mL doxycycline (DOX) treatment in OCI-AML5 cells expressing a DOX-inducible shRNA against RUNX1. (H) OCI-AML5 i-sh-RUNX1 cells were induced with the indicated concentrations of DOX for 96 hours. At the end of treatment, cells were stained with propidium iodide, and the percent nonviable cells were determined according to flow cytometry. Columns, mean of 3 experiments; bars, standard error of the mean. *P < .05, **P < .01 relative to no-DOX treatment. (I) Bioluminescent imaging of mice after infusion of OCI-AML5/GFP-Luc cells transduced with DOX-inducible RUNX1-shRNA (i-sh-RUNX1) and treated daily with 10 mg/kg of DOX. Mice were also infused with OCI-AML5/GFP-Luc cells that had been ex vivo treated with DOX for 72 hours, then treated daily with 10 mg/kg of DOX to further suppress leukemia engraftment. The box plots beneath show the total bioluminescent flux for the mice treated with DOX vs no DOX over 5 weeks of treatment. (J) The survival of the mice in the 3 cohorts is represented by a Kaplan-Meier survival plot. n.s., not significant.