Fig. 2

BCL2L2 is a transcriptional target of WT1. RNA sequencing was performed on U937 cells transduced with retroviral MSCV-WT1 or negative control MSCV-NC vectors. a Heatmap representation of genes regulated by WT1. Shown is BCL2L2, which is positively regulated by WT1. b Scatter plots of genes exhibiting twofold upregulation (red plots) or twofold downregulation (green plots) upon expression of WT1. c and d Gene set enrichment analysis (GSEA) was performed comparing transcriptional profiles of U937 cells with overexpression of WT1 in comparison to NC control. The enrichment score plot shown corresponds to the regulation of myeloid cell differentiation genes (c) and hematopoietic cell lineage genes (d). The NES and P-values are shown. e The transcriptional expression of BCL2L2 was measured in U937 cells, which were transduced with MSCV-WT1 or control MSCV-NC. f The protein expression of BCL2, BCL2L1, BCL2L2, BAK, BAD, and BAX were determined in U937 cells transduced with MSCV-WT1 or control MSCV-NC. g The transcript of bcl2l2 was measured in BM GFP⁺ blasts isolated from MLL-AF9-induced murine leukemia with knockdown of wt1 (n = 4) and control nc (n = 4). h The protein levels of wt1 and bcl2l2 were assessed in BM GFP⁺ blasts isolated from MLL-AF9-induced murine leukemia with knockdown of wt1 (n = 2) and control nc (n = 2). i Putative BCL2L2 promoter, including GC-rich sequences, was constructed into the pGL3-basic vector. Both firefly and renilla luciferase activities were measured in 293T cells, which were transduced with different concentrations of pCMV-WT1, as well as pGL3 vector carrying BCL2L2 promoter. pRL-SV40 vector containing the renilla luciferase gene was transduced into 293T cells for internal control. Histograms illustrate firefly luciferase activities normalized to renilla. Normalized luciferase activity of NC-transfected cells was arbitrarily set to 1.0