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Results: 5

1.
FIGURE 1.

FIGURE 1. From: G-protein-coupled Receptor Agonist BV8/Prokineticin-2 and STAT3 Protein Form a Feed-forward Loop in Both Normal and Malignant Myeloid Cells.

BV8 induces STAT3 activation in both malignant and normal myeloid cells. A, Western blotting showing phospho-STAT3 (p-STAT3) and STAT3 protein amounts in KG1 and U937 cells treated with human BV8 (200 ng/ml) at the indicated time points. B, Western blotting showing increased expression of phospho-STAT3 (p-STAT3) proteins in tumor-primed, splenic CD11b+ myeloid cells upon treatment with BV8 (100 ng/ml) for 6 or 16 h. Cells were enriched from splenocytes of B16 tumor-bearing mice; mean ± S.E., n = 3.

Hong Xin, et al. J Biol Chem. 2013 May 10;288(19):13842-13849.
2.
FIGURE 3.

FIGURE 3. From: G-protein-coupled Receptor Agonist BV8/Prokineticin-2 and STAT3 Protein Form a Feed-forward Loop in Both Normal and Malignant Myeloid Cells.

BV8-induced STAT3 activation requires JAK2. A, Western blotting showing expression levels of indicated proteins in splenic CD11b+ cells enriched from B16 tumor-bearing mice with Jak2+/+ or Jak2−/− hematopoietic cells or in mouse endothelial cells. Cells were treated with BV8 for 6 h. p-JAK2, phospho-JAK2; p-STAT3, phospho-STAT3. B, Western blotting to determine expression levels of indicated proteins in splenic CD11b+ cells, which were enriched from naive C57BL/6 mice, treated with AZD1480 (1 μm) or DMSO for 1 h, and subsequently stimulated with BV8 (100 ng/ml) for the indicated times. C, real-time RT-PCR to measure Bv8 mRNA levels in splenic CD11b+ cells freshly isolated from Renca tumor-bearing mice treated with AZD1480 or vehicle for 6 days (left); **, p < 0.01 (mean ± S.E., n = 3). Western blotting showing expression levels of indicated proteins (right). D, Bv8 mRNA levels in splenic CD11b+ cells isolated from B16 tumor-bearing mice with Jak2+/+ and Jak2−/− hematopoietic cells *, p < 0.05 (mean ± S.E., n = 3).

Hong Xin, et al. J Biol Chem. 2013 May 10;288(19):13842-13849.
3.
FIGURE 5.

FIGURE 5. From: G-protein-coupled Receptor Agonist BV8/Prokineticin-2 and STAT3 Protein Form a Feed-forward Loop in Both Normal and Malignant Myeloid Cells.

Inhibiting BV8 expression in myeloid leukemia cells reduces STAT3 activity and tumor growth. A, BV8 gene expression (left) and phospho-STAT3 (p-STAT3) protein expression levels (right) in KG1 cells transduced with control or BV8 shRNA were analyzed by real-time RT-PCR and Western blotting, respectively (mean ± S.E., n = 3). B, representative photo images (left) show the differential growth of abdominal tumors in mice implanted with control or BV8 shRNA-expressing KG1 cells. Right, bar graph showing tumor volumes in each group at the end of studies; for control shRNA group, and for Bv8 shRNA group, *, p < 0.05 (mean ± S.E., n = 3). C, Western blotting showing expression levels of indicated proteins in tumors. Each lane represents a sample from an individual mouse. D, left, representative images of immunofluorescent staining showing CD31+ blood vessels in xenograft tumors. Right, quantifications of CD31+ blood vessels per field (200×) are also shown *, p < 0.05 (mean ± S.E., n = 3). E, a proposed model for a BV8-STAT3 feed-forward loop in both normal and malignant myeloid cells.

Hong Xin, et al. J Biol Chem. 2013 May 10;288(19):13842-13849.
4.
FIGURE 4.

FIGURE 4. From: G-protein-coupled Receptor Agonist BV8/Prokineticin-2 and STAT3 Protein Form a Feed-forward Loop in Both Normal and Malignant Myeloid Cells.

Silencing BV8 expression reduces myeloid leukemia cell viability and expression of proliferation/survival and angiogenic genes. A–C, BV8-STAT3 regulates leukemia cell proliferation/survival. Cell viability assay was performed in KG1 or U937 cells expressing either control or BV8 shRNA cultured in RPMI 1640 medium with 5% FBS for 48 h (A); KG1 or U937 cells cultured in RPMI 1640 medium (200 ng/ml BV8, 1% FBS) for 48 h (B); or KG1 cells expressing either control or STAT3 shRNA cultured in RPMI 1640 medium (200 ng/ml BV8, 1% FBS) for 48 h (C). Data are generated from six independent experiments with triplicates for each experiment; *, p < 0.05; **, p < 0.01. D, flow cytometric analysis of cell cycle phase in KG1 cells expressing control or BV8 shRNA. Cells in the culture medium were stained with propidium iodide solution. Representative histograms show the cell cycle distribution in KG1 cells. Mean percentages of cell counts in each cycle are shown under the histogram as well as in the graph; mean ± S.D.; n = 2, *, p < 0.05; **, p < 0.01. E, a graph showing the percentage of apoptotic KG1 cells. KG1 cells were serum-starved for 48 h before staining with annexin V solution. Data are representative of three independent experiments; *, p < 0.05. F, real-time RT-PCR showing mRNA levels of STAT3 target genes in KG1 or U937 cells stably expressing control or BV8 shRNA; mean ± S.E., n = 3; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Hong Xin, et al. J Biol Chem. 2013 May 10;288(19):13842-13849.
5.
FIGURE 2.

FIGURE 2. From: G-protein-coupled Receptor Agonist BV8/Prokineticin-2 and STAT3 Protein Form a Feed-forward Loop in Both Normal and Malignant Myeloid Cells.

STAT3 regulates BV8 expression in both malignant and normal myeloid cells. A, BV8 and STAT3 gene expression levels in stable cell lines (KG1 and U937) expressing indicated shRNA were analyzed by real-time RT-PCR. B, left, real-time RT-PCR showing BV8 gene expression in KG1 or U937 cells expressing control or STAT3 shRNA; **, p < 0.01. Right, immunoprecipitation (IP) by BV8 antibodies followed by Western blotting (IB) showing expression levels of KG1 cell-derived BV8 protein. C, BV8 mRNA levels in G-CSF-treated KG1 cells expressing control or STAT3 shRNA shown by real-time RT-PCR; *, p < 0.05. D, upper, BV8 mRNA levels in KG1 cells transfected with control plasmid or plasmid encoding STAT3C; *, p < 0.05. Lower, Western blotting to detect FLAG-tagged STAT3C protein in cells transfected with the indicated plasmids. E, ChIP assays showing STAT3 binding to the BV8 promoter in KG1 cells stably expressing control or STAT3 shRNA. Immunoprecipitated DNA fragments were detected by PCR (left) or quantitative real-time RT-PCR (right). Bottom left, a graph showing the relative intensity of DNA bands in the PCR gel (top), which were obtained by ImageJ software and then plotted as a percentage of the band intensity in each sample to the corresponding input (100%) (bottom left). Bottom right, data from real-time PCR assay were first calculated as (DNA amount in anti-STAT3 immunoprecipitation minus DNA in IgG immunoprecipitation)/(DNA in input), and then compared with the data from the anti-STAT3 antibody immunoprecipitation sample of STAT3 shRNA-expressing cells, which is designated as 1; mean ± S.E., n = 3; *, p < 0.05. F, real-time RT-PCR measuring Bv8 mRNA levels in splenic CD11b+ cells freshly isolated from tumor-bearing mice with Stat3+/+ or Stat3−/− hematopoietic cells (mean ± S.E., n = 3).

Hong Xin, et al. J Biol Chem. 2013 May 10;288(19):13842-13849.

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