![Figure 2. MafB and PU.1 induce alternative macrophage or DC phenotype and function in myeloblasts. E26ts21 MafB–expressing myeloblasts (MafB) were induced to differentiate for 48 hours by temperature shift to 42°C (which releases a vMyb-imposed differentiation block), E26ts21-PUER (PU.1), and E26ts21-control (ctrl) were induced by 1 μM βE treatment. (A,D) May-Grünwald-Giemsa (MGG) staining of cytocentrifuged cells (objective lens ×40). (B,E) FACS analysis for the chicken monocyte/macrophage marker 47.83. (C,F) Cells with DC (▪) or macrophage (MΦ) morphology (▦) were counted for a total of at least 100 cells in 3 independent PUER (C) and MafB (F) clones from 2 separate experiments. (G) Mixed lymphocyte reaction (MLR) of myeloid cells differentiated by PU.1 or MafB activation. Ctrl, PU.1, and MafB clones were induced (ind.) or not induced (–) to differentiate, treated with 25 μg/mL mitomycin C, and incubated in duplicates in the absence (–) or presence (spleno.) of 105 monocyte-depleted allogenic splenocytes at different stimulating cell–splenocyte ratios for 5 days. [3H]-thymidine incorporation was measured after 16 hours of labeling. (H) Stimulating activity of 6 independent clones in 3 different experiments was analyzed and is expressed as proliferation index at a stimulating cell–splenocyte ratio of 1. (I) Phagocytic capacity of myeloid cells differentiated by PU.1 or MafB activation. Induced ctrl, PU.1, and MafB clones were incubated for 2 hours with phycoerythrin (PE)–conjugated latex beads and analyzed by FACS. Phagocytic activity before and after induction of differentiation was determined for 6 independent clones in 3 different experiments and expressed as the product of percent positive cells (as a measure of phagocytosing cells) and their mean fluorescence intensity (as a measure of the number of phagocytosed beads). In H-I, ▦ indicates uninduced cells; ▪, induced. Error bars indicate the standard error of the mean. Images in panels A and D were acquired using a Leica DMiL optical microscope equipped with a 40×/1.0 objective lens and a Leica MPS30 camera. Contrast and exposure were enhanced with Adobe Photoshop 5.0 software (Adobe, San Jose, CA) using equal treatment on all panels.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/105/7/10.1182_blood-2004-04-1448/6/zh80070576480002.jpeg?Expires=1723141317&Signature=MTHzytDg4m6BPYX5J8i1~UBUKZvchRXeMroVxKsh8E4qGq-m9LuQTvN79gvLQp6MbF9PtQ6tbMzZ~Si4Xg06LFGJxH3nQPHNdhhbB5ul33cZIXy6ps8~pI866NZxv6R-SSFiQHcbOyinCysBLPudEz-2iDWb3MLpNYFjtXRVpgf2bRSWthNCtqiQHIYdPFMDSoAYcq-k4LhCZi4Xj2JY1t7mSvRAmC1Gg95i-N-0pTb80JbkyNfi1uzt8NS2MCpOXnv~layx60kRIHLCDk8OQmKoEcvJPVgUaQWWYyQBMnjeIRSpoQuZzJ5YQ~BR6j4r8BNqtH5Uhz69DENR-dGuJA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
MafB and PU.1 induce alternative macrophage or DC phenotype and function in myeloblasts. E26ts21 MafB–expressing myeloblasts (MafB) were induced to differentiate for 48 hours by temperature shift to 42°C (which releases a vMyb-imposed differentiation block), E26ts21-PUER (PU.1), and E26ts21-control (ctrl) were induced by 1 μM βE treatment. (A,D) May-Grünwald-Giemsa (MGG) staining of cytocentrifuged cells (objective lens ×40). (B,E) FACS analysis for the chicken monocyte/macrophage marker 47.83. (C,F) Cells with DC (▪) or macrophage (MΦ) morphology (▦) were counted for a total of at least 100 cells in 3 independent PUER (C) and MafB (F) clones from 2 separate experiments. (G) Mixed lymphocyte reaction (MLR) of myeloid cells differentiated by PU.1 or MafB activation. Ctrl, PU.1, and MafB clones were induced (ind.) or not induced (–) to differentiate, treated with 25 μg/mL mitomycin C, and incubated in duplicates in the absence (–) or presence (spleno.) of 105 monocyte-depleted allogenic splenocytes at different stimulating cell–splenocyte ratios for 5 days. [3H]-thymidine incorporation was measured after 16 hours of labeling. (H) Stimulating activity of 6 independent clones in 3 different experiments was analyzed and is expressed as proliferation index at a stimulating cell–splenocyte ratio of 1. (I) Phagocytic capacity of myeloid cells differentiated by PU.1 or MafB activation. Induced ctrl, PU.1, and MafB clones were incubated for 2 hours with phycoerythrin (PE)–conjugated latex beads and analyzed by FACS. Phagocytic activity before and after induction of differentiation was determined for 6 independent clones in 3 different experiments and expressed as the product of percent positive cells (as a measure of phagocytosing cells) and their mean fluorescence intensity (as a measure of the number of phagocytosed beads). In H-I, ▦ indicates uninduced cells; ▪, induced. Error bars indicate the standard error of the mean. Images in panels A and D were acquired using a Leica DMiL optical microscope equipped with a 40×/1.0 objective lens and a Leica MPS30 camera. Contrast and exposure were enhanced with Adobe Photoshop 5.0 software (Adobe, San Jose, CA) using equal treatment on all panels.
