Figure 1.
Genotyping of JAK2V617F/p53-/- mice and J53Z1 cells.
Genomic DNAs isolated from mouse tails and cultured cells. JAK2V617F transgene, wild type 53, mutant p53, and endogenous mouse Jak2 were PCR-amplified with specific primers described in Materials and Methods. The PCR products were resolved on 3% agarose gels and visualized by ethidium bromide staining. HCD-57, a previously established mouse erythroleukmia cell line, was analyzed for comparison.
Figure 2.
Growth curves of J53Z1 cells in suspension culture.
J53Z1 cells were grown in IMDM medium supplemented with 20% fetal bovine serum. Cell numbers were counted by using flow cytometery as described in Materials and Methods. Error bars denote standard deviation (n = 3).
Figure 3.
Normal growing J53Z1 cells were attached to glass slides by cytospin and then subjected to Wright-Giemsa staining. HCD-57 erythroid leukemia cells were stained for comparison. Photos were taken with a 100x objective lens.
Figure 4.
Flow cytometric analysis of CD71 and TER-119 expression on J53Z1 cells.
J53Z1, HCD-57, and normal mouse bone marrow cells were labeled with anti-CD71 and TER-119 monoclonal antibodies or with a nonspecific isotype control mouse IgG before flow cytometric analysis.
Figure 5.
Real time PCR assays of gene expression in J53Z1 cells.
Expressions of indicated genes were analyzed by real time PCR using specific PCR primers shown in the top panel. Data represent relative mRNA levels (mean±SD, n = 3) normalized to mouse GAPDH which was defined as 1000. HCD-57 and normal mouse bone cells were analyzed for comparison. *P<0.001 in reference to normal bone marrow cells.
Table 1.
Surface Markers on J53Z1 Cells.
Figure 6.
Response of J53Z1 cells to growth factors.
J53Z1 cells were serum-starved for 5 hr and then stimulated with 10 Units/ml EPO or 50ng/ml SCF for 10 min. Cell extracts were analyzed for activation of indicated signaling proteins by using phospho-specific antibodies. Equal protein loading was demonstrated by blotting with anti-actin.
Figure 7.
Development of erythroleukemia phenotypes in mice receiving implantation of J53Z1 cells.
Cultured J53Z1 cells (1×106) were implanted into 12-week-old wild type C57Bl/6 mice through retro-orbital injections. Upper panel. Red blood cells, spleen, and liver were analyzed 2 to 4 weeks after implantation. Error bars denote standard deviation (n≥4), *P<0.001. Lower panel. Paraffin sections of spleen and liver from representative control and J53Z1-implanted mice were subjected to H&E staining. Note the loss of normal tissue architecture and infiltration of densely stained erythroleukemia cells in tissues from J53Z1-transplanted mice. Photos were taken with a 40x objective lens.
Figure 8.
Inhibition of J53Z1 cells by selective protein kinase inhibitors.
J53Z1 cells were cultured in the presence of various concentrations of indicated protein kinase inhibitors. Top and middle panels. Cell viability was assessed by XTT assays after 72 hr of incubation. Control experiments were performed in the presence of 0.1% DMSO. Error bars denote standard deviation (n = 3). *P<0.001 in reference to control. Note that MV-4-11 cells were analyzed for comparison (middle panel). Bottom panel. Apoptosis assays were performed with J53Z1 cells after 24 hr of incubation with 0.2 µM of ruxolitinib or AZD1480. Cells were stained with FITC-annexin V and propidium iodide. Percentages of annexin V-positive cells are indicated.