Fig. 2: PU.1 mutants show impaired differentiation potential.

a PU.1 mutants have impaired transactivation potential. F9 cells (no endogenous c-Jun) and CV-1 cells (expressing c-Jun) were transfected with a PU.1-responsive human M-CSFR promoter-luciferase reporter and either PU.1 alone or in combination with c-Jun to test coactivation potential. Mean of values of three independent experiments (n = 3) ±SD are shown. b PU.1 mutants fail to induce macrophage differentiation in a PU.1-deficient progenitor cell line. Wright-Giemsa staining of wild-type PU.1 (PU.1-wt) and mutant PU.1 (Q202L, Y252N, and F203Y) transduced 503 cells at day 14 cultured in differentiation medium. Magnification was ×1000. In contrast to only vector-transduced cells (Vector), 503 cells transduced with wild-type PU.1 differentiate into monocytes. The arrow (PU.1-wt 2-1) points to a cell being phagocytosed by a macrophage, as well as mature granulocytes with segmented nuclei (PU.1-wt 2–2 and 2–3). 503 cells transduced with the PU.1 mutants showed immature myeloid blasts similar to mock-transduced or parental 503 cells. Three independent experiments were performed with representative images shown. c Cell surface expression of the late macrophage marker F4/80 correlates with morphological signs of differentiation. 503 cells were transduced with wild-type (PU.1-wt) or mutant PU.1(Q202L, Y252N and F203Y), or with empty retroviral expression vector (Vector). Infected (EGFP⁺) cells were sorted, cultured for 10 days in differentiation medium and analyzed by flow cytometry for expression of F4/80 and EGFP expression. Protein extracts of the sorted cells were used for western blots analysis to assess PU.1 expression levels. (Fig. 2c, lower left panel). Three independent experiments were performed with representative images shown. Source data for the blot can be found in Supplemental Data 1.