MGMT downregulation in GFI1-36N cells due to low levels of NDRG1. (A) Fold change expression of Mgmt in murine GFI1-36S- and GFI1-36N-MLL-AF9 leukemic BM cells at different time points after actinomycin D (10 μg/mL) treatment. Mgmt level was normalized to Hprt and to the untreated controls. n = 3; mean ± SD. (B) NDRG1 protein level in GFI1-36S- and GFI1-36N-MLL-AF9 leukemic BM cells (proteomic). n = 4; mean ± SD. (C) Ndrg1 expression (normalized read counts; RNA-seq) of murine leukemic GFI1-36S- and GFI1-36N-MLL-AF9 BM cells. n = 3; mean ± SD. (D) Ndrg1 gene expression measured in GFI1-36S- and GFI1-36N-MLL-AF9 BM cells by RT-PCR. GFI1-36S: n = 3 and GFI1-36N: n = 3; mean ± SD. (E) Published GFI1-ChIP-seq data sets showing the Ndrg1 gene and its regulatory elements with the possible binding sides of GFI1 (red square) at regulatory elements of Ndrg1. (F) GFI1-ChIP-quantitative PCR of the Ndrg1 upper regulatory elements of murine GFI1-36S and GFI1-36N leukemic BM cells. Gapdh and Runx1 were used as a control (right). (G) Comparison between the GFI1 binding motif from the Jasper database (top) and the consensus motif found using find individual motif occurrence (FIMO) at sites occupied by GFI1 in 21 genes differentially expressed in granulocyte/monocyte progenitors s from GFI1-36N or -36S animals. (H) Ndrg1 expression (RNA-seq) in murine leukemic GFI1-36S- and GFI1-36N-MLL-AF9 cells after treatment with 50 μg/mL TMZ for 20 hours and without. Normalized read counts of treated samples were normalized to the untreated samples. n = 3; mean ± SD. (I) NDRG1 protein level was analyzed by immunoblotting in BM cells from GFI1-36S and GFI1-36N leukemic mice without and with TMZ (50 μg/mL) treatment for 24 hours. ∗P < .05; ∗∗∗P < .001; ∗∗∗∗P < .0001.