Figure 5.

Expression of CrmA or FADD-DN inhibits colony formation by hemopoietic progenitor cells. Fetal liver cells from E14 C57BL/6 embryos were infected with retroviruses encoding FLAG-CrmA-GFP, FLAG–FADD-DN–GFP, or GFP alone (control vector) for 48 hours in the presence of IL-6, mSCF, TPO, and Flk ligand (Flk)L. After infection, cells were sorted for GFP expression and cultured for 7 days in agar with SCF, IL-3, and EPO. (A) Compared with GFP vector–expressing cells, hemopoietic progenitor cells expressing FLAG-CrmA or FLAG–FADD-DN formed very few colonies. Colonies were viewed under a Leica DMLIL microscope (Leica, Wetzlar, Germany) using a 20× objective (0.0-0.4 NA) and photos were taken with an Axiocam camera and processed using Axiovision software (both from Zeiss, Hallbergmoos, Germany). (B) The numbers of colonies are shown graphically with data representing mean ± SD from 6 to 9 independent experiments for each vector. CFU indicates colony-forming units. (C) Similar experiments were performed with fetal liver cells infected with retroviruses encoding GFP alone (vector), wt FLAG-CrmA, mutated FLAG-CrmA (CrmAmt), which does not inhibit caspase-8, dominant-interfering catalytically inactive caspase-8 (FLAG–caspase-8–DN) or dominant-interfering catalytically inactive caspase-9 (FLAG–caspase-9–DN). (D) Retroviruses encoding GFP alone (vector), FLAG–FADD-DN, a mutant of FLAG–FADD-DN that is unable to bind to the intracellular region of Fas²⁶ or the cytoplasmic region of Fas (FLAG-cFas), were also used. The numbers of colonies are shown graphically with data representing arithmetic mean ± SD from 6 to 9 independent experiments. (E) Freshly isolated fetal liver cells, not cultured and not enriched for progenitor cells, were plated in agar culture in the presence of Z-Val-Ala-Asp-(ome)-fluoromethyl-ketone (Z-VAD-fmk) at the specified concentrations together with optimal cytokines. Colonies were counted after 7 days in culture, and numbers represent means ± SDs from 3 separate experiments.