Effects of palmostatin B on CFU-GM and blast colony growth. (A-B) CFU-GM were grown from fetal liver cells expressing N-Ras^G12D, K-Ras^G12D, N-Ras^{G12D, KHVR}, and K-Ras^{G12D, NHVR} at 0 and 0.1 ng/mL GM-CSF in the presence palmostatin B. As in Figure 1, the data presented are from 3 independent experiments. Asterisks indicate statistically significant reductions in colony growth compared with untreated cells that were transduced with the same vector and plated in parallel: *P < .05; ***P < .0005. Only cells infected with MSCV vectors encoding proteins containing the N-Ras HVR are sensitive to treatment. (C) Confocal imaging of differentiated macrophages from GFP⁺ fetal liver cells after treatment with 10μM palmostatin B for 15 minutes. Proteins containing the N-Ras HVR show reduced localization at the plasma membrane by palmostatin B treatment. 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. (D) Effects of palmostatin B on cytokine-independent CFU-GM growth from the bone marrows of 3-month-old Mx1-Cre; LSL-Nras^G12D/+, Mx1-Cre; LSL-Kras^G12D/+, Mx1-Cre; LSL-Nras^G12D/G12D mice as well as from 6-month-old Mx1-Cre, LSL-Nras^G12D/G12D mice. One mouse of each genotype was analyzed in 2 independent experiments. Asterisks indicate statistically significant reductions in CFU-GM growth compared with untreated cells of the same genotype: *P < .05; **P < .005; ***P < .0005. (E) Recipient mice that were transplanted with Mx1-Cre, LSL-Nras^G12D and Mx1-Cre, LSL-Kras^G12D AML cells died of aggressive leukemia (leukocyte counts > 100 000/mm³). Blast colony growth was assessed from bone marrow cells plated in 10 ng/mL GM-CSF with or without palmostatin B. The growth of CFU-GM colonies from a WT mouse was compared with blast colony growth from the same Kras mutant AML and from 2 Nras AMLs in 2 independent experiments. Asterisks indicate statistically significant reductions in CFU-GM growth compared with untreated cells from the same mice: *P < .05; **P < .005.