Critical Roles of Myc-ODC Axis in the Cellular Transformation Induced by Myeloproliferative Neoplasm-Associated JAK2 V617F Mutant
Figure 5
JAK2 (V617F)-induced cell proliferation and transformation require the expression of c-Myc.
VF/EpoR cells were infected with retrovirus harboring shRNA against firefly luciferase (control) or c-Myc. (A) Transduced VF/EpoR cells were cultured without Epo for 12 hr. Whole cell lysates were immunoblotted (IB) with anti-c-Myc antibody, anti-ODC antibody or anti-β-actin antibody. (B) ODC mRNA was analyzed by quantitative real-time PCR. Data are the mean ± S.D. of the relative expression levels in three independent experiments. (C) Transduced VF/EpoR cells were cultured without Epo for 3 days. Viable cells were counted and shown in the graph. Results are the mean ± S.D. of three independent experiments. (D) Transduced VF/EpoR cells were cultured without Epo for 24 hr. Cells were then fixed, treated with propidium iodide (PI) and subjected to FACS analysis. (E-I) Transduced VF/EpoR cells were s.c. injected into nude mice (1×107 cells/mice). (E) Nude mice were photographed 16 days post-inoculation. Arrows indicate tumors in nude mice. (F, G) Sixteen days post-inoculation, three mice were sacrificed. Morphological changes of the spleen and liver are shown in the photograph. The weights of the tumor, liver, and spleen were measured and plotted on the graph. * and ** indicate significant differences p<0.05 and p<0.01, respectively. (H) Sixteen days after inoculation, H&E staining was performed (magnification: ×100). (I) Mouse survival was monitored daily for 30 days post-inoculation (n = 10).