PMC

(A) FACS analysis of bone marrow samples from a wild type (wt-bone marrow) and two moribund MN1-mice to determine the size of the CMP, MEP and GMP populations. GFP⁻ and GFP⁺ gates were used for wild type or MN1-bone marrow cells, respectively. (B) Giemsa staining of leukemic bone marrow cells of the same mice as in A showing their morphology. Arrows indicate different myeloid cell types.

(A) Comparison of in vivo leukemogenic activity of MN1 and Δ1260–1320 transduced bone marrow after transplantation in 10 lethally irradiated mice each. (B) FACS GFP analysis of the peripheral blood (PB) of mice that received transplants of BM expressing MN1 or the indicated MN1 mutants at the indicated time points after transplantation. WBC: White blood cells. (C) Kaplan-Meier survival plots of mice transplanted with bone marrow cells transduced with each indicated retrovirus.

(A, B, C) Pair wise comparison of CMP/GFP⁺ cells transduced with MN1, Δ12–228, Δ18–458 retrovirus with CMP/GFP⁺ cells transduced with empty vector (mock). (D) Venn diagram showing the number of probe sets that are differentially expressed in the pair wise comparison in A, B and C. (E) Hierarchical clustering of 502 transcripts with >2-fold differential expression in MN1 CMP/GFP⁺ cells and two bulk leukemic MN1-bone marrow (Leuk #1, #2) as compared with CMP/GFP⁺ cells transduced with empty vector (GFP). Heat map indicates expression relative to the mean in standard deviation units.

(A) Schematic representations of MN1 deletion mutants. Mutants depicted in blue do cause leukemia, while mutants in red do not. Leukemia types generated by each mutant are indicated in the Figure. Stippled lines indicate the size of MN1 deletions. (B) Comparison of Kaplan-Meier survival curves of mice receiving transplants of bone marrow transduced with the indicated MN1 deletion mutants. (C) Histological analysis of a bone marrow sample of a representative leukemic mouse in each indicated group. Images were taken at 400X using an Olympus BX41 microscope, equipped with an Insight 2 Mega-pixel Color Mosaic camera, employing SPOT advanced imaging software. H&E = hematoxilin & eosin staining, GFP = green fluorescent protein staining, MPO = myeloperoxidase staining.

(A) FACS analysis showing the distribution of MEP, CMP and GMP populations in lineage depleted mouse bone marrow HSPC that were transduced with retroviruses expressing GFP alone (mock), MN1, or the indicated MN1-deletion mutants. Freshly isolated bone marrow cells were used to set up the gates. Figure shows one representative results from 3 independent experiments. (B) Mouse HSPCs transduced with indicated retroviruses were subjected to a second lineage depletion 96 hrs after transduction (Day-0) and cells were cultured for an additional 2 days (Day-2). FACS analysis shows Mac1 and Gr1 expression of unsorted cells at Day-0 and Day-2 of culture. (C) FACS analysis showing the Sca1 and c-Kit expression of the GFP (+) cells in panel B.

(A) Schematic representation of MN1 and MN1-deletion mutant proteins. (B) Western blot analysis of GFP-sorted U937 stable cell lines overexpressing the indicated proteins. Figure shows immunoblotting results of anti-MN1 antibodies recognizing either the N-terminus (top left panel) or C-terminus of the protein (top right panel) and anti-human ACTIN antibody (bottom right panel). The commassie-blue stained image of the PAGE gel (bottom left panel; CB) and a pan-actin blot (bottom right panel; ACTIN) are shown as loading controls. (C) Immuno-cytochemical analysis of the above cells using the same MN1-antibodies (red). Blue shows nuclear staining with 4,6-diamidino-2-phenylindole (DAPI). Images were captured with an Olympus BX-50 microscope (equipped with UPlanFL 40×/0.75 numerical apertures with a SPOT camera and SPOT Advanced Imaging software of Diagnostic Instruments. The original magnification was x400. (D, E) FACS analysis showing CD11b expression of indicated U937 stable cell lines 3 days after treatment with vehicle, vitamin D₃ (Vit-D3) or ATRA (mean ± SEM of duplicates; *P<.05, ***P<.001, ^#not significant).

(A) Periodic GFP analysis was performed at indicated times of liquid cultures using FACS. GFP percentage at Day-0 of analysis (4 days after the last transduction) in each sample was set to 1 and the results of all indicated time points were calculated relative to Day-0. Graph shows one representative analysis of three independent experiments. (B) Methylcellulose assays showing colony numbers after serial replating (1 to 5) of linage-depleted mouse HSPCs transduced with the indicated retroviruses (mean ± SEM of duplicates). (C) May-Grunwald/Giemsa stained images of cytospins of the cells in panel B (after the first methylcellulose culture). (D, E) Differential-count of cytospins is shown in panel C. A total of 100 cells were counted in each sample. The maturation pool includes neutrophils and metamyelocytes whereas the proliferation pool comprises myelocytes and promyelocytes. The images in panels C and D were captured with an Olympus BX41 microscope, equipped with SPOT Insight Color Mosaic 2 MP camera and SPOT imaging software (original magnification x1000).