Fig. 5.

Triad1 knockdown or Shp2 activation increased ubiquitination-induced and cytokine-induced proliferation in Mll-Ell-transduced murine bone marrow cells. Murine myeloid progenitor cells were transduced with vectors to express Mll-Ell, Mll-Af9, Triad1, Triad1-specific shRNAs, E76K-Shp2, C463S-Shp2, or relevant vector controls. Cells were analyzed with or without G-CSF-differentiation. a Triad1 knockdown decreased the Mll-Ell-induced increase in total protein ubiquitination. Western blots of cell lysates were probed with ubiquitinated proteins (Ub), Triad1, Ell, or Gapdh (loading control) antibodies. b Mll-Ell increased Triad1 expression in myeloid progenitor cells throughout granulocyte differentiation. Significant differences indicated by *, **, ***, #, ##, ###, or &. Bars in these graphs are means and error bars ± SEM. c Triad1 knockdown increased cytokine hypersensitivity of Mll-Ell or Mll-Af9 expressing murine myeloid progenitor cells. Proliferation was determined by 3H-thymidine incorporation during GM-CSF-induced proliferation. Significant differences in incorporation at common GM-CSF doses are indicated by * or **. d Constitutive Shp2 activity increased cytokine hypersensitivity in Mll-Ell-expressing myeloid progenitor cells. Significant differences in ³H-thymidine incorporation at common GM-CSF doses indicated by * or **. e Dominant negative Shp2 decreased cytokine hypersensitivity in Mll-Ell-expressing myeloid progenitor cells. Significant differences in ³H-thymidine incorporation at common GM-CSF doses indicated by *. f Triad1 impairs Fgf2-induced proliferation of myeloid progenitor cells. Cells were analyzed for proliferation in response to various amounts of Fgf2. Significant differences in ³H-thymidine incorporation at common Fgf2 doses indicated by * or **. Circles or crosses represent means and error bars ± SEM