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

Journal: Frontiers in Immunology

Article Title: Icaritin Induces Anti-tumor Immune Responses in Hepatocellular Carcinoma by Inhibiting Splenic Myeloid-Derived Suppressor Cell Generation

doi: 10.3389/fimmu.2021.609295

Figure Lengend Snippet: Icaritin reduces the accumulation of HSPCs in the spleen of Hepa mice. Spleen weight and total cell number from orthotopic (A) and subcutaneous (B) Hepa mice. Numbers of splenic LSK and LK cells from orthotopic (C) and subcutaneous (D) Hepa mice. Differences between groups were examined for statistical significance by Student's t -test. Data were pooled from two experiments and n = 8 mice per group. * P < 0.05, ** P < 0.01, *** P < 0.001. HSPCs, hematopoietic stem and progenitor cells; Hepa, Hepa1-6 cells; ICT, icaritin; Veh, Vehicle; LSK, Lin lo/− Sca-1 + c-Kit hi ; LK, Lin lo/− Sca-1 − c-Kit hi .

Article Snippet: In brief, human CD34 + cells were isolated from the cord blood mononuclear cells using a direct CD34 progenitor cell isolation kit (Miltenyi Biotec), and cultured in hematopoietic stem cell expansion media (StemSpan SFEM; Stem Cell Technologies) supplemented with 100 ng/mL stem cell factor, 100 ng/mL Fms-like tyrosine kinase 3, 100 ng/mL thrombopoietin, and 20 ng/mL IL-3 (R&D Systems) for 9 days ( ).

Techniques:

Journal: Frontiers in Immunology

Article Title: Icaritin Induces Anti-tumor Immune Responses in Hepatocellular Carcinoma by Inhibiting Splenic Myeloid-Derived Suppressor Cell Generation

doi: 10.3389/fimmu.2021.609295

Figure Lengend Snippet: Icaritin inhibits the generation of human PMN-MDSCs in vitro . Freshly isolated human CD34 + cells from cord blood mononuclear cells were cultured in hematopoietic stem cell expansion media for 8–10 days. The expanded cells were cultured with combined IL-6 and G-CSF in complete medium with 0.1% DMSO (vehicle) or icaritin (2.5 μM) for 3 days. (A) Flow cytometric analysis of CD115 and HLA-DR expression on CD14 + or CD15 + cells. (B) Frequencies of PMN-MDSCs (CD15 + CD115 + ) and M-MDSCs (CD14 + HLA-DR − ). Differences between groups were analyzed using one-way ANOVA, and corrected by Dunnett's test. Data are representative of three experiments. ** P < 0.01. Veh, Vehicle; ICT, Icaritin.

Article Snippet: In brief, human CD34 + cells were isolated from the cord blood mononuclear cells using a direct CD34 progenitor cell isolation kit (Miltenyi Biotec), and cultured in hematopoietic stem cell expansion media (StemSpan SFEM; Stem Cell Technologies) supplemented with 100 ng/mL stem cell factor, 100 ng/mL Fms-like tyrosine kinase 3, 100 ng/mL thrombopoietin, and 20 ng/mL IL-3 (R&D Systems) for 9 days ( ).

Techniques: In Vitro, Isolation, Cell Culture, Expressing