Figure 4
Figure 4. Sustained AKT signaling in myr-AKT mice causes depletion of LSK cells and progenitors and increased apoptosis. (A) Left bar graph: Percentage GFP of BM transduced with myr-AKT-GFP or MIG control retrovirus on the day of tail vein injection. Results are the mean of data from 3 independent transplantation experiments. Right bar graphs: Percentage GFP in recipient BM and spleen (Spln) in MIG or myr-AKT mice killed at 6 to 8 weeks after transplantation. Error bars represent SEM. (B) Decrease in percentage GFP+ cells in the LSK and progenitor compartments of myr-AKT BM. BM of control MIG transplantation mice or diseased myr-AKT transplantation mice killed at 6 to 8 weeks after transplantation was stained with a lineage antibody cocktail, goat anti–rat antibody, and then Sca1 and c-Kit to distinguish the LSK and progenitor populations. The percentage of GFP+ cells in each population is shown. (C) Competitive engraftment of BM transduced with myr-AKT or MIG control retrovirus. BM was transduced with myr-AKT or MIG retrovirus and then injected into mice as previously described. Recipient mice were killed at 2 weeks after transplantation, and percentage GFP in the BM was quantified by flow cytometry. The fold change in percentage GFP in the BM was determined as: percentage GFP in recipient BM/percentage GFP in donor BM. The values for MIG controls were normalized to 1 for the analysis. (D) Apoptosis analysis of the LSK and progenitor compartments in BM from MIG or diseased myr-AKT mice killed at 6 to 8 weeks after transplantation. Apoptosis analysis of the LSK and mixed progenitor populations from myr-AKT mice. BM cells were gated on GFP+ LSK and Lin−c-kit+Sca1− (Prog) cells. Representative annexin V vs propidium iodide plots are shown for these populations. (E) Methylcellulose plating assays of BM cells from myr-AKT and MIG control mice, killed at 6 to 8 weeks after transplantation. Bar graphs reveal the total number of colonies seen at each round of replating every 7 days. (F) Methylcellulose plating assays of spleen cells from myr-AKT and MIG control mice, killed at 6 to 8 weeks after transplantation. Bar graphs represent the total number of colonies seen at each round of replating every 7 days.

Sustained AKT signaling in myr-AKT mice causes depletion of LSK cells and progenitors and increased apoptosis. (A) Left bar graph: Percentage GFP of BM transduced with myr-AKT-GFP or MIG control retrovirus on the day of tail vein injection. Results are the mean of data from 3 independent transplantation experiments. Right bar graphs: Percentage GFP in recipient BM and spleen (Spln) in MIG or myr-AKT mice killed at 6 to 8 weeks after transplantation. Error bars represent SEM. (B) Decrease in percentage GFP+ cells in the LSK and progenitor compartments of myr-AKT BM. BM of control MIG transplantation mice or diseased myr-AKT transplantation mice killed at 6 to 8 weeks after transplantation was stained with a lineage antibody cocktail, goat anti–rat antibody, and then Sca1 and c-Kit to distinguish the LSK and progenitor populations. The percentage of GFP+ cells in each population is shown. (C) Competitive engraftment of BM transduced with myr-AKT or MIG control retrovirus. BM was transduced with myr-AKT or MIG retrovirus and then injected into mice as previously described. Recipient mice were killed at 2 weeks after transplantation, and percentage GFP in the BM was quantified by flow cytometry. The fold change in percentage GFP in the BM was determined as: percentage GFP in recipient BM/percentage GFP in donor BM. The values for MIG controls were normalized to 1 for the analysis. (D) Apoptosis analysis of the LSK and progenitor compartments in BM from MIG or diseased myr-AKT mice killed at 6 to 8 weeks after transplantation. Apoptosis analysis of the LSK and mixed progenitor populations from myr-AKT mice. BM cells were gated on GFP+ LSK and Lin−c-kit+Sca1− (Prog) cells. Representative annexin V vs propidium iodide plots are shown for these populations. (E) Methylcellulose plating assays of BM cells from myr-AKT and MIG control mice, killed at 6 to 8 weeks after transplantation. Bar graphs reveal the total number of colonies seen at each round of replating every 7 days. (F) Methylcellulose plating assays of spleen cells from myr-AKT and MIG control mice, killed at 6 to 8 weeks after transplantation. Bar graphs represent the total number of colonies seen at each round of replating every 7 days.

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