GFI1-36N leukemic cells are highly susceptible to TMZ treatment. (A) Functional Mgmt assays from murine GFI1-36S and GFI1-36N Lin– cells (45-206 cells per sample) and (B) GFI1-36S and GFI1-36N MLL-AF9 BM cells from mice that received transplantation (115-223 cells per sample). Cells were treated with 100 μg/mL TMZ and at different time points after treatment O6MeG level was analyzed with immunofluorescence. The ACAS program was used for the evaluation. (C) Cell viability of murine GFI1-36S and GFI1-36N MLL-AF9 BM cells measured by MTT assay after treatment with different TMZ concentrations for 48 hours, and IC50 values were calculated; mean ± SD. (D) Schematic experimental setup of the colony-forming unit (CFU) assays. (E) Murine GFI1-36S and GFI1-36N Lin– cells and (F) MLL-AF9 BM cells from mice were plated for 14 days in methylcellulose medium with the addition of 50 μg/mL TMZ or as a control dimethyl sulfoxide (DMSO). The colony number of the treated samples was calculated relative to the control. n = 3; mean ± SD. (G) CFU assay was performed with malignant BM cells from transgenic GFI1-36Sx and GFI1-36NxNUP98-HOXD13 mice. Cells were treated with 50 μg/mL TMZ and as a control with DMSO. The colony number after 14 days in culture of the treated samples was calculated relative to the control. n = 2, each triplicate, mean ± SD. (H) MGMT protein level was measured by immunoblotting in BM of GFI1-36S and GFI1-36N leukemic mice without and with TMZ (50 μg/mL) treatment for 24 hours. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. AFU, arbitrary fluorescence units (O6MeG/4′,6-diamidino-2-phenylindole); IC50, 50% inhibitory concentration.
