Functional analysis of N-Ras^G12D mutant proteins. (A) CFU-GM growth of GFP⁺ fetal liver cells expressing WT N-Ras, N-Ras^G12D, and N-Ras^G12D HVR mutant proteins over a range of GM-CSF concentrations. The data are shown as percentage of maximal growth (left panel) and the absolute number of colonies (right panel) for each construct. The data presented are from 3 independent experiments. Asterisks on the right panel indicate statistically significant differences in colony growth: *P < .05; **P < .005; ***P < .0005. Cytokine-independent CFU-GM growth was only observed in cells expressing N-Ras^{G12D, SSDD} or N-Ras^G12D, and was significantly lower for the SSDD mutant. For statistical analyses, the number of CFU-GM colonies that formed in cells expressing WT N-Ras in the presence of a saturating concentration of GM-CSF (10 ng/mL) was compared with all 3 mutants. Cells expressing N-Ras^{G12D, SSDD} or N-Ras^G12D formed significantly more colonies, whereas cells expressing N-Ras^{G12D, C181S} formed significantly fewer. (B) Confocal imaging of macrophages differentiated from GFP⁺ fetal liver cells. Note that the SSDD mutant protein accumulates in the Golgi and that the C181S mutant is absent from the plasma membrane. The confocal images were acquired on the Zeiss LSM 510 NLO Meta using the Plan-APOCHROMAT 63×/1.4 aperture oil objective. Images were taken on live cells grown on Lab-Tek chambered coverglass w/cvr at 25°C. We used GFP as the fluorochrome, and fluorescent signals were detected using photomultiplier tubes. We used the acquisition software LSM 510 and no further manipulation of the images was performed. (C) Biochemical analysis of cultured GFP⁺ fetal liver cells differentiated into macrophages in vitro. The cells were deprived of serum overnight and stimulated with 10 ng/mL GM-CSF for 20 minutes. The 3 G12D mutant proteins accumulate in the GTP-bound conformation, and both total Ras expression and ERK activation are severely attenuated by the C181S substitution. (D) CFU-GM growth of fetal liver cells expressing WT N-Ras and WT N-Ras with the C181S mutation over a range of GM-CSF concentrations. The data presented are from 3 independent experiments.