Treatment with ODC inhibitor, DFMO, significantly inhibited tumorigenesis induced by JAK2 (V617F).

<p>Ba/F3 cells were infected with empty virus (-) and retrovirus encoding JAK2 V617F mutant and EpoR. Transduced Ba/F3 cells were s.c. injected into nude mice (1×10⁷ cells/mice) and then given either standard water (H₂O) or water containing 1% DFMO for 10 days. (A) Nude mice were photographed 16 days post-inoculation. Arrows indicate tumors in nude mice. (B, C) 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 in the graph. * and ** indicate significant differences p<0.05 and p<0.01, respectively. (D) Sixteen days after inoculation, sections of the liver were stained with H&E (magnification: ×100). (E) Mouse survival was monitored daily for 30 days post-inoculation (n = 10).</p

Inhibition of GSK-3β inhibited downregulation of c-Myc and apoptosis.

<p>(A) Transduced Ba/F3 cells were treated with DMSO (0.1%) or the indicated concentrations of GSK-3β inhibitor in the absence of IL-3 for 18 hr. The viability of these cells was determined by the trypan blue exclusion method. Results are the mean ± S.D. of three independent experiments. (B-E) Transduced Ba/F3 cells were treated with DMSO (0.1%) or GSK-3β inhibitor (0.1 µM) for 18 hr. (B) Whole cell lysates were immunoblotted (IB) with anti-phospho-c-Myc antibody (Thr58), anti-c-Myc antibody or anti-β-actin antibody. (C) The expression amounts of c-Myc and its mutants were normalized with the protein amount of β-actin, and the quantified ratios of c-Myc and its mutants (T58A, In373) are shown in the graph. Results are the mean ± S.D. of three independent experiments. (D) Total RNA was prepared and mRNA of ectopic c-Myc and its mutants (T58A, In373) was detected by quantitative real time-PCR. GAPDH mRNA was analyzed as an internal control. Data are the mean ± S.D. of the relative expression levels in three independent experiments. (E) DNA was isolated from cells and subjected to agarose gel electrophoresis. (F, G) Ba/F3 cell lines were infected with empty virus (−), retrovirus encoding JAK2 mutant c-HA (V617F) and retroviruses encoding wild-type EpoR c-Flag (WT) or EpoR mutant c-Flag (Y479F). Transduced Ba/F3 cells were incubated without IL-3 for 12 hr. (F) Whole cell lysates were immunoblotted (IB) with anti-phospho-JAK2 antibody (Y1007/1008), anti-phospho-GSK-3β antibody (S9), anti-GSK-3β antibody, anti-c-Myc antibody, anti-Bcl-XL antibody, anti-HA antibody, anti-Flag antibody or anti-β-actin antibody. (G) c-Myc mRNA and Bcl-XL mRNA were analyzed by quantitative real-time PCR. GAPDH mRNA was evaluated as an internal control. Data are the mean ± S.D. of the relative expression levels in three independent experiments.</p

ODC inhibitor, DFMO, significantly induced G0/G1 arrest of transformed Ba/F3 cells by JAK2 (V617F).

<p>(A, B) EpoR cells, WT/EpoR cells and VF/EpoR cells were treated with the indicated concentrations of DFMO in the presence or absence of Epo (5 U/mL) for 3 days. (A) Viable cells were counted and shown in the graph. Results are the mean ± S.D. of three independent experiments. (B) The viability of these cells was evaluated by the trypan blue exclusion method. Results are the mean ± S.D. of three independent experiments.</p

c-Myc T58A mutant conferred growth-factor independence on Ba/F3 cells.

<p>(A) Schematic diagram of wild-type (c-Myc), T58A and In373 mutants of c-Myc. (B-H) Ba/F3 cells were infected with empty virus (−) or retrovirus encoding wild-type c-Myc (c-Myc) or two c-Myc mutants (T58A, In373). (B) Transduced cells were incubated without IL-3 (2 ng/mL) for 6 hr and whole cell lysates were immunoblotted (IB) with anti-c-Myc antibody or anti-β-actin antibody. (C, D) Transduced Ba/F3 cells were cultured with/without IL-3 (2 ng/mL) for 12 hr. (C) ODC mRNA was analyzed by quantitative real-time PCR. GAPDH mRNA was analyzed as an internal control. Data are the mean ± S.D. of the relative expression levels in three independent experiments. (D) Whole cell lysates were immunoblotted (IB) with anti-ODC antibody or anti-β-actin antibody. (E) Viable transduced Ba/F3 cells and VF/EpoR cells were counted in the presence and absence of IL-3 (2 ng/mL) for 3 days. Data are the mean ± S.D. of the relative expression levels in three independent experiments. (F) Transduced Ba/F3 cells were left untreated or stimulated with IL-3 (2 ng/mL) for 24 hr. The viability of these cells was determined by the trypan blue exclusion method. Results are the mean ± S.D. of three independent experiments. (G) Transduced cells were cultured without IL-3 (2 ng/mL) for 24 hr. Cells were then fixed, treated with propidium iodide (PI) and subjected to FACS analysis. (H) Transduced Ba/F3 cells were cultured with/without IL-3 (2 ng/mL) for 24 hr. DNA was isolated from cells and subjected to agarose gel electrophoresis.</p

JAK2 (V617F) induced expression of c-Myc through STAT5 activation.

<p>(A–E) Ba/F3 cell lines were infected with empty virus (−), retroviruses encoding wild-type JAK2 c-HA (WT), JAK2 mutant c-HA (V617F) and EpoR c-Flag as indicated in each figure. (A) WT/EpoR cells and VF/EpoR cells were cultured without Epo for 12 hr, and then total RNAs were prepared from each cell line. The enhancement of gene expression induced by JAK2 (V617F) was determined by DNA array. The ratio of the altered gene expressions in VF/EpoR cells was calculated by dividing the amount of each gene in VF/EpoR cells by their amount in WT/EpoR cells. (B, C) A series of Ba/F3 cells expressing the described genes was cultured untreated or stimulated with Epo (5 U/mL) for 12 hr. (D, E) A series of Ba/F3 cells expressing the indicated genes were cultured in the absence of Epo, and treated with DMSO (0.1%) or AG490 (30 µM) for 12 hr. (F) HEL cells were treated with DMSO (0.1%) or AG490 (10, 20, 30 µM) for 24 hr. (G, H) VF/EpoR cells were infected with retrovirus harboring shRNA against firefly luciferase (control) or STAT5. (I, J) Ba/F3 cells were infected with empty virus (−), retrovirus encoding wild-type STAT5 (WT) or the constitutively active mutant of STAT5 (1*6). The cells were cultured without Epo for 12 hr. (B, E, H, J) The mRNA expression of c-Myc and ODC was analyzed by quantitative real-time PCR. GAPDH mRNA was analyzed as an internal control. Data are the mean ± S.D. of the relative expression levels in three experiments. (C, D, F, G, I) Whole cell lysates were immunoblotted (IB) with anti-c-Myc antibody, anti-ODC antibody, anti-HA antibody, anti-EpoR antibody, anti-phospho-JAK2 antibody (Y1007/1008), anti-phospho-STAT5 antibody (Y694), anti-STAT5 antibody or anti-β-actin antibody.</p

c-Myc T58A mutant significantly induced tumor formation in nude mice.

<p>Ba/F3 cells were infected with empty virus (-) and retrovirus encoding wild-type c-Myc (c-Myc) or two c-Myc mutants (T58A, In373). Transduced Ba/F3 cells and VF/EpoR cells were s.c. injected into nude mice (1×10⁷ cells/mice). (A) Nude mice were photographed 18 days post-inoculation. Arrows indicate tumors in nude mice. (B, C) Eighteen days post-inoculation, three mice were sacrificed. The morphology of the liver and spleen were photographed. The weights of the spleen and liver were measured and shown in the graphs. * indicates significant difference p<0.01 (n = 3). (D) Sixty days after inoculation, sections of the spleen and liver were stained with hematoxylin and eosin (magnification: ×100). (E) Mouse survival was monitored daily for 100 days post-inoculation (n = 10).</p

JAK2 (V617F)-induced cell proliferation and transformation require the expression of c-Myc.

<p>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).</p

List of Ba/F3-derived cell lines analyzed in this study.

a<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052844#pone-0052844-g002" target="_blank">Fig. 2A</a> shows structures of c-Myc mutants, T58A and In373.</p>b<p>c-Myc expression was tested under the absence of Epo stimulation.</p>c<p>ND means “Not Detected”.</p>d<p>Tumorigenesis was tested by the transplantation of cells into nude mice.</p

Coffee extract inhibits adipogenesis in 3T3-L1 preadipocyes by interrupting insulin signaling through the downregulation of IRS1

<div><p>Although epidemiological data have indicated that a strong negative association exists between coffee consumption and the prevalence of obesity-associated diseases, the molecular mechanisms by which coffee intake prevents obesity-associated diseases has not yet been elucidated. In this study, we found that coffee intake significantly suppressed high-fat diet (HFD)-induced metabolic alternations such as increases in body weight and the accumulation of adipose tissue, and up-regulation of glucose, free fatty acid, total cholesterol and insulin levels in the blood. We also found that coffee extract significantly inhibited adipogenesis in 3T3-L1 preadipocytes. In the early phase of adipogenesis, 3T3-L1 cells treated with coffee extract displayed the retardation of cell cycle entry into the G2/M phase called as mitotic clonal expansion (MCE). Coffee extract also inhibited the activation of CCAAT/enhancer-binding protein β (C/EBPβ) by preventing its phosphorylation by ERK. Furthermore, the coffee extract suppressed the adipogenesis-related events such as MCE and C/EBPβ activation through the down-regulation of insulin receptor substrate 1 (IRS1). The stability of the IRS1 protein was markedly decreased by the treatment with coffee extract due to proteasomal degradation. These results have revealed an anti-adipogenic function for coffee intake and identified IRS1 as a novel target for coffee extract in adipogenesis.</p></div

Effects of coffee extract on expression of IRβ, IRS1, and IRS2 in 3T3-L1 cells.

<p>(A) 3T3-L1 cells were treated with 5% (v/v) coffee extract for 1 hr, and then stimulated with MDI for the indicated periods. Cell lysates were immunoprecipitated (IP) with an anti-IRS1 antibody, followed by immunoblotting (IB) with an anti-phospho-tyrosine (PY) antibody or anti-IRS1 antibody. Whole cell lysates were immunoblotted with an anti-β-actin antibody. The phosphorylation level of IRS1 was normalized with the expression level of IRS1 or the expression level of β-actin. The relative phosphorylation of IRS1 is shown in the graphs. Values are the mean ± S.D. of three independent experiments. **<i>p</i><0.01 vs. control cells. (B, C) 3T3-L1 cells were treated with 5% (v/v) coffee extract for the indicated periods. (B) Cell lysates were immunoblotted with an anti-IRβ antibody, anti-IRS1 antibody, anti-IRS2 antibody, or anti-β-actin antibody. The expression level of IRβ, IRS1, and IRS2 was normalized with the protein amount of β-actin. The relative expression level of IRβ, IRS1, and IRS2 is shown in the graphs. Values are the mean ± S.D. of three independent experiments. (C) Total RNA was prepared and the mRNA expression of <i>Irβ</i>, <i>Irs1</i>, and <i>Irs2</i> was analyzed using quantitative real-time PCR. <i>β-actin</i> mRNA was analyzed as an internal control. Values are the mean ± S.D. of three independent experiments. **<i>p</i><0.01 vs. untreated cells.</p