Figure 2
Figure 2. Inclusion of the PHD fingers in MLL-AF9 leads to hematopoietic cell differentiation and reduced Hoxa9 expression. (A) Transduced mouse bone marrow was grown in liquid culture under G418 selection and in the presence of SCF and IL-3. A significant growth advantage was observed in MLL-AF9 and MLL-PHD#1-AF9–transduced cells. (B) MLL-AF9 and MLL-PHD#1-AF9 fusion proteins were detected by Western blot in cell lines generated from transduced bone marrow. N/S indicates Nonspecific band serving as loading control. (C) Morphology of transduced bone marrow was examined by Wright-Giemsa staining. Inclusion of 2 or more PHD fingers induced a more differentiated phenotype. Scale bars = 50 μm. N/A indicates not applicable. Micrographs were obtained using Olympus BX51 microscope, 100×/1.40 PLAN APO oil immersion objective lens, Olympus DP70 camera, Olympus DP70 acquisition software, and Olympus DP controller software. (D) Hoxa9 expression was examined using real-time PCR and cDNA from cells collected after the first round of colony assays. Significant suppression of Hoxa9 is detected after transduction with fusion constructs containing 2 of more PHD fingers. Error bars indicate SD from experiment performed in triplicate. N/D indicates not detected. (E) Luciferase assay showing significant transactivation by MLL-AF9 and MLL-PHD1-AF9. Assays were performed in transiently transfected 293 cells in serum starved growth conditions. A schematic of the Hoxa9-luciferase reporter used in the assay is shown. One of 3 representative experiments is shown. #P < .001 determined using Student t test. (F) Chromatin immunoprecipitation assay was performed in transiently transfected 293 cells. Immunoprecipitations were performed with Flag antibodies or normal rabbit IgG. Binding was detected by real-time PCR with the P1 primer set (▶, ◀) amplifying the promoter of HOXA9. Statistical significance was determined using Student t test. One of 3 representative experiments is shown. Similar results were obtained using a separate primer set amplifying the HOXA9 promoter.

Inclusion of the PHD fingers in MLL-AF9 leads to hematopoietic cell differentiation and reduced Hoxa9 expression. (A) Transduced mouse bone marrow was grown in liquid culture under G418 selection and in the presence of SCF and IL-3. A significant growth advantage was observed in MLL-AF9 and MLL-PHD#1-AF9–transduced cells. (B) MLL-AF9 and MLL-PHD#1-AF9 fusion proteins were detected by Western blot in cell lines generated from transduced bone marrow. N/S indicates Nonspecific band serving as loading control. (C) Morphology of transduced bone marrow was examined by Wright-Giemsa staining. Inclusion of 2 or more PHD fingers induced a more differentiated phenotype. Scale bars = 50 μm. N/A indicates not applicable. Micrographs were obtained using Olympus BX51 microscope, 100×/1.40 PLAN APO oil immersion objective lens, Olympus DP70 camera, Olympus DP70 acquisition software, and Olympus DP controller software. (D) Hoxa9 expression was examined using real-time PCR and cDNA from cells collected after the first round of colony assays. Significant suppression of Hoxa9 is detected after transduction with fusion constructs containing 2 of more PHD fingers. Error bars indicate SD from experiment performed in triplicate. N/D indicates not detected. (E) Luciferase assay showing significant transactivation by MLL-AF9 and MLL-PHD1-AF9. Assays were performed in transiently transfected 293 cells in serum starved growth conditions. A schematic of the Hoxa9-luciferase reporter used in the assay is shown. One of 3 representative experiments is shown. #P < .001 determined using Student t test. (F) Chromatin immunoprecipitation assay was performed in transiently transfected 293 cells. Immunoprecipitations were performed with Flag antibodies or normal rabbit IgG. Binding was detected by real-time PCR with the P1 primer set (▶, ◀) amplifying the promoter of HOXA9. Statistical significance was determined using Student t test. One of 3 representative experiments is shown. Similar results were obtained using a separate primer set amplifying the HOXA9 promoter.

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