Journal: Nature Communications

Article Title: MOZ/ENL complex is a recruiting factor of leukemic AF10 fusion proteins

doi: 10.1038/s41467-023-37712-5

Figure Lengend Snippet: a Structures required for AF10 fusion-mediated myeloid transformation. Various AF10 fusion constructs were examined to assess their transforming ability of myeloid progenitors. HA-tag (indicated by red triangles) was fused to MTM and MTMT constructs. FLAG-tag (indicated by blue triangles) was fused to other AF10 fusion constructs. Dotted lines indicate protein-protein interaction. A schema of a myeloid progenitor transformation assay is shown at the top. Hoxa9 expression normalized to Gapdh in first-round colonies (left) is shown as the relative value of CALM-AF10 (arbitrarily set at 100%) (mean of two biological replicates). Colony-forming ability at the third- and fourth-round passages (right) is shown with error bars (mean ± SD of biological replicates, n ≥ 3). b Association of NES-AF10 fusion with ENL in the presence or absence of DOT1L. IP-western blotting (WB) analyses of the chromatin fraction of HEK293T cells [the parental clone or a DOT1L-knockout clone (dDOT1L)] transiently expressing the FLAG-tagged (indicated as f) NES-AF10´ fusion (fNES-AF10´) construct and Xpress-tagged (indicated as x) ENL (xENL) were performed. Co-purification of ENL was observed only in the presence of DOT1L. c Leukemogenesis by NES-ENL fusion in vivo. Various AF10 fusion-derivatives including NES-ENL were transduced to c-Kit-positive hematopoietic progenitors and transplanted into syngeneic mice. Primary NES-ENL leukemia cells were harvested from the bone marrow (BM) and transplanted into recipient mice. d Hierarchical clustering analysis of RNA-seq profiles of AF10 fusion-ICs. Normalized count data in various AF10 fusion-ICs was clustered using R ward D2 method. Source data are provided as a Source Data file.

Article Snippet: Human hematopoietic stem cell—CD34 + cells the from fetal liver (purchased from Cell Applications, INC.) were pre-cultured in Hematopoietic Stem Cell Culture Medium (Cell Applications, INC.) for 4 days and plated in methylcellulose-based medium (MethoCult TM H4435 Enriched, STEMCELL Technologies) in the presence of designated drugs for 6 days.

Techniques: Transformation Assay, Construct, FLAG-tag, Expressing, Western Blot, Knock-Out, Copurification, In Vivo, RNA Sequencing