Journal: Journal of Cancer

Article Title: PFKFB4 promotes angiogenesis via IL-6/STAT5A/P-STAT5 signaling in breast cancer.

doi: 10.7150/jca.66773

Figure Lengend Snippet: Figure 4. IL-6 induces STAT5 phosphorylation in HUVEC cells. A) The Western blot analysis showed that conditioned medium collected from MDA-MB-231/PFKFB4 and T47D/PFKFB4 cells induced IL-6R expression of HUVEC cells. β-actin was used as a loading control. B) The representative immunofluorescent photomicrographs illustrated that conditioned medium collected from MDA-MB-231/PFKFB4 (upper panel) and T47D/PFKFB4 (lower panel) augmented IL-6R immunostaining (green) of HUVEC cells. The nuclei (blue) were stained using DAPI. C) The Western blot analysis showed that conditioned medium collected from MDA-MB-231/PFKFB4 and T47D/PFKFB4 cells induced STAT5 phosphorylation, STAT5A and CD31 expression of HUVEC cells, but not STAT1 or STAT3 phosphorylation. STAT1, STAT3, STAT5B, and β-actin were used as a loading control. D) The Western blot analysis showed that knocking down of STAT5A, but not STAT5B expression in HUVEC, abolished STAT5 phosphorylation promoted by the conditioned medium collected from MDA-MB-231/PFKFB4 and T47D/PFKFB4 cells. β-actin was used as a loading control. E) The Western blot analysis showed that anti-IL-6 treatment of HUVEC blocked STAT5 phosphorylation prompted by the conditioned medium collected from MDA-MB-231/PFKFB4 and T47D/PFKFB4 cells. β-actin was used as a loading control.

Article Snippet: Western blot was performed using the following antibodies: PFKFB4 (1:1000, ab137785, Abcam), IL-6 (1:1000, WL02841, Wanleibio), IκBα (phosphor S36) (1:1000, ab133462, Abcam), IκBα (1:1000, ab76429, Abcam), p-NF-κBp65 ser536 (1:1000, WL02169, Wanleibio), NF-κBp65 (1:1000, sc-398442, Santa Cruz), β-actin (1:2000, sc-47778, Santa Cruz), Lamin A/C (1:1000, 2032, CST), α-Tubulin (1:1000, 2144, CST), CD31 (1:1000, WL03674, Wanleibio), IL-6R (1:1000, WL02737, Wanleibio), p-STAT1 (Y701) (1:1000, 7649, CST), p-STAT3 (Y705) (1:1000, 9145, CST), p-STAT5 (Y694) (1:1000, 9359, CST), STAT3 (1:1000, 9139, CST) , STAT1 (1:1000, sc-592, Santa Cruz), STAT5A (1:1000, sc-1081, Santa Cruz), STAT5B (1:1000, sc-1656, Santa Cruz).

Techniques: Phospho-proteomics, Western Blot, Expressing, Control, Immunostaining, Staining

Journal: Journal of Cancer

Article Title: PFKFB4 promotes angiogenesis via IL-6/STAT5A/P-STAT5 signaling in breast cancer.

doi: 10.7150/jca.66773

Figure Lengend Snippet: Figure 5. 5-MPN treatment inhibits PFKFB4-induced angiogenesis signaling in vitro. A) The representative photomicrographs of 3 independent tube formation assays showed that 5-MPN treatment of MDA-MB-231 and T47D cells inhibited PFKFB4-induced HUVEC tube formation compared to the DMSO/PFKFB4 group. This bar graph shows the quantification of tube formation assay that illustrates 5-MPN treatment of MDA-MB-231 and T47D cells significantly decreased HUVEC tube formation versus the DMSO/PFKFB4 group (16.67±0.88 versus 6.00±1.16; 17.00±1.53 versus 5.00±1.16, respectively; ** p<0.01, *** p<0.001). B) The Western blot analysis showed that 5-MPN treatment of MDA-MB-231 and T47D cells diminished PFKFB4-induced NF-κB phosphorylation and IL-6 expression. β-actin was used as a loading control. C) The Western blot analysis showed that 5-MPN treatment of MDA-MB-231 and T47D cells diminished PFKFB4-induced STAT5 phosphorylation and STAT5A, IL-6R, CD31 expression in HUVEC cells. β-actin was used as a loading control.

Article Snippet: Western blot was performed using the following antibodies: PFKFB4 (1:1000, ab137785, Abcam), IL-6 (1:1000, WL02841, Wanleibio), IκBα (phosphor S36) (1:1000, ab133462, Abcam), IκBα (1:1000, ab76429, Abcam), p-NF-κBp65 ser536 (1:1000, WL02169, Wanleibio), NF-κBp65 (1:1000, sc-398442, Santa Cruz), β-actin (1:2000, sc-47778, Santa Cruz), Lamin A/C (1:1000, 2032, CST), α-Tubulin (1:1000, 2144, CST), CD31 (1:1000, WL03674, Wanleibio), IL-6R (1:1000, WL02737, Wanleibio), p-STAT1 (Y701) (1:1000, 7649, CST), p-STAT3 (Y705) (1:1000, 9145, CST), p-STAT5 (Y694) (1:1000, 9359, CST), STAT3 (1:1000, 9139, CST) , STAT1 (1:1000, sc-592, Santa Cruz), STAT5A (1:1000, sc-1081, Santa Cruz), STAT5B (1:1000, sc-1656, Santa Cruz).

Techniques: In Vitro, Tube Formation Assay, Western Blot, Phospho-proteomics, Expressing, Control

Journal: Journal of Cancer

Article Title: PFKFB4 promotes angiogenesis via IL-6/STAT5A/P-STAT5 signaling in breast cancer.

doi: 10.7150/jca.66773

Figure Lengend Snippet: Figure 6. 5-MPN treatment inhibits PFKFB4-induced angiogenesis in vivo. A) Scheme for the in vivo experiment with results shown in panels B, C, D and E. B) Photographs of xenograft MDA-MB-231 tumors formed in NOD/SCID mice harvested on Day 30. The photographs qualitatively indicate that ectopic expression of PFKFB4 increased tumor size versus the MCS group, whereas, 5-MPN treatment inhibited PFKFB4-induced growth of tumor size. C) Growth curve of xenograft MDA-MB-231 tumors formed in NOD/SCID mice. This graph demonstrates that ectopic expression of PFKFB4 significantly increased tumor size versus the MCS group (1364±36.05 versus 1897+44.31, respectively; *** p<0.001), whereas, 5-MPN treatment significantly inhibited PFKFB4-induced growth of tumor size (1897±44.31 versus 746.6±27.59, respectively; *** p<0.001). Tumor volume = length x width x width/2. D) The tumor weight of xenograft MDA-MB-231 tumors formed in NOD/SCID mice. The dot plot demonstrates that ectopic expression of PFKFB4 significantly increased tumor weight versus the MCS group (0.71±0.06 versus 1.22±0.11, respectively; **p<0.01), whereas, 5-MPN treatment significantly inhibited PFKFB4-induced growth of tumor size (1.22±0.11 versus 0.55±0.08, respectively; ***p<0.001). E) The representative micrographs of IL-6, P-NF-κB, IL-6R, CD31, STAT5A, and P-STAT5 immunocytochemical staining in xenograft MDA-MB-231 tumors. Ectopic expression of PFKFB4 increased immunocytochemical staining of above-mentioned molecules versus the MCS group, whereas, 5-MPN treatment inhibited PFKFB4-induced immunocytochemical staining of above-mentioned molecules. Scale bar= 50 µm.

Article Snippet: Western blot was performed using the following antibodies: PFKFB4 (1:1000, ab137785, Abcam), IL-6 (1:1000, WL02841, Wanleibio), IκBα (phosphor S36) (1:1000, ab133462, Abcam), IκBα (1:1000, ab76429, Abcam), p-NF-κBp65 ser536 (1:1000, WL02169, Wanleibio), NF-κBp65 (1:1000, sc-398442, Santa Cruz), β-actin (1:2000, sc-47778, Santa Cruz), Lamin A/C (1:1000, 2032, CST), α-Tubulin (1:1000, 2144, CST), CD31 (1:1000, WL03674, Wanleibio), IL-6R (1:1000, WL02737, Wanleibio), p-STAT1 (Y701) (1:1000, 7649, CST), p-STAT3 (Y705) (1:1000, 9145, CST), p-STAT5 (Y694) (1:1000, 9359, CST), STAT3 (1:1000, 9139, CST) , STAT1 (1:1000, sc-592, Santa Cruz), STAT5A (1:1000, sc-1081, Santa Cruz), STAT5B (1:1000, sc-1656, Santa Cruz).

Techniques: In Vivo, Expressing, Staining

Journal: Journal of Cancer

Article Title: PFKFB4 promotes angiogenesis via IL-6/STAT5A/P-STAT5 signaling in breast cancer.

doi: 10.7150/jca.66773

Figure Lengend Snippet: Figure 7. A proposed model for PFKFB4-induced angiogenesis. PFKFB4 promotes angiogenesis via IL-6/STAT5A/P-STAT5 signaling in breast cancer. PFKFB4 ectopic expression in breast cancer cells elevates lactate levels in the culture medium which initiates NF-κB activation and nuclear translocation. NF-κB within the nucleus binds to the IL-6 promoter region and then enhances IL-6 expression. The resultant IL-6 expression boosts IL-6R and CD31 (a vascular differentiation marker) expression in endothelial cells. Consequently, it appears that STAT5A/P-STAT5 (but not STAT3) is the pivotal signaling molecules involved in the angiogenic process.

Article Snippet: Western blot was performed using the following antibodies: PFKFB4 (1:1000, ab137785, Abcam), IL-6 (1:1000, WL02841, Wanleibio), IκBα (phosphor S36) (1:1000, ab133462, Abcam), IκBα (1:1000, ab76429, Abcam), p-NF-κBp65 ser536 (1:1000, WL02169, Wanleibio), NF-κBp65 (1:1000, sc-398442, Santa Cruz), β-actin (1:2000, sc-47778, Santa Cruz), Lamin A/C (1:1000, 2032, CST), α-Tubulin (1:1000, 2144, CST), CD31 (1:1000, WL03674, Wanleibio), IL-6R (1:1000, WL02737, Wanleibio), p-STAT1 (Y701) (1:1000, 7649, CST), p-STAT3 (Y705) (1:1000, 9145, CST), p-STAT5 (Y694) (1:1000, 9359, CST), STAT3 (1:1000, 9139, CST) , STAT1 (1:1000, sc-592, Santa Cruz), STAT5A (1:1000, sc-1081, Santa Cruz), STAT5B (1:1000, sc-1656, Santa Cruz).

Techniques: Expressing, Activation Assay, Translocation Assay, Marker

Journal: Blood Cancer Journal

Article Title: Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia

doi: 10.1038/bcj.2013.56

Figure Lengend Snippet: Flow cytometric gating strategy adopted to identify CD33+/CD34+ precursor cells. A representative JMML patient is showed. Mononuclear cells were initially gated to exclude debris and residual granulocytes by physical parameters ( a ); all myeloid cells were selected by their reactivity to anti-CD33 antibody ( b ); myeloid precursors were then identified as CD33+/CD34+ double positive cells ( c ). CD33+/CD34+ cells were further checked for their negativity to anti-CD14 antibody ( d ) and low expression of CD45 ( e ), as features of myeloid precursor cells. p-STAT5 response was then measured on these selected cells by dual SSC/STAT5 cytogram ( f ). In panel ( e ), only CD33+/CD34+/CD14− gated cells are shown. In panel ( f ), only CD33+/CD34+/CD14−/CD45low gated cells are shown.

Article Snippet: Samples were incubated with anti-phospho-STAT5 (p-STAT5) Alexa 488 (Y694, clone 47, BD Biosciences) or isotype IgG 1 k Alexa 488 (clone MOPC-21, BD Biosciences), anti-CD33 PE (clone P67.6, BD Biosciences), anti-CD34 APC (clone 8G12, BD Biosciences), anti-CD45 PerCP (clone 2D1, BD Biosciences) and anti-CD38 PE-Cy7 (clone HB7, BD Biosciences) antibodies.

Techniques: Expressing

Journal: Blood Cancer Journal

Article Title: Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia

doi: 10.1038/bcj.2013.56

Figure Lengend Snippet: Training set samples assessed the best threshold. The p-STAT5 responses were measured at each GM-CSF concentration in the training set of samples (11 JMMLs and 23 controls). The best dose to distinguish JMML from non-JMML samples was identified at 0.1 ng/ml of GM-CSF ( P <0.0001). p-STAT5-positive cells (%) were quantified by scaling the maximum % of p-STAT5+ cells at 100 and the unstimulated p-STAT5+ cells to 0.

Article Snippet: Samples were incubated with anti-phospho-STAT5 (p-STAT5) Alexa 488 (Y694, clone 47, BD Biosciences) or isotype IgG 1 k Alexa 488 (clone MOPC-21, BD Biosciences), anti-CD33 PE (clone P67.6, BD Biosciences), anti-CD34 APC (clone 8G12, BD Biosciences), anti-CD45 PerCP (clone 2D1, BD Biosciences) and anti-CD38 PE-Cy7 (clone HB7, BD Biosciences) antibodies.

Techniques: Concentration Assay

Journal: Blood Cancer Journal

Article Title: Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia

doi: 10.1038/bcj.2013.56

Figure Lengend Snippet: Representative flow cytometric contour plots of p-STAT5 response in CD33+/CD34+ cells. Dual SSC/STAT5 cytograms from a JMML (upper panels) and a control (lower panels) are shown. Contour plots are referred to CD33+/CD34+ cells identified by gating strategy described in . For each dose of GM-CSF, the raw percentage of responding p-STAT5-positive cells is shown. Response to stimulation at each GM-CSF dose was then quantified by scaling the maximum percentage of p-STAT5+ cells at 100 and the unstimulated p-STAT5+ cells to 0. According to this criteria, calculated p-STAT5 responses are indicated in parenthesis for each stimulation dose.

Article Snippet: Samples were incubated with anti-phospho-STAT5 (p-STAT5) Alexa 488 (Y694, clone 47, BD Biosciences) or isotype IgG 1 k Alexa 488 (clone MOPC-21, BD Biosciences), anti-CD33 PE (clone P67.6, BD Biosciences), anti-CD34 APC (clone 8G12, BD Biosciences), anti-CD45 PerCP (clone 2D1, BD Biosciences) and anti-CD38 PE-Cy7 (clone HB7, BD Biosciences) antibodies.

Techniques:

Journal: Blood Cancer Journal

Article Title: Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia

doi: 10.1038/bcj.2013.56

Figure Lengend Snippet: Comparison of p-STAT5-positive cells (%) induced by 0.1 ng/ml of GM-CSF in the validation series comprising 11 JMML samples (central box plot), 24 controls (left box plot) and 7 samples from patients with other diseases mimicking JMML at presentation (right box plot). The discriminating threshold (17.17% as assessed in the training set) is indicated. The bold line inside each box plot indicates the median level, while the upper and lower lines indicate the maximum and minimum observed values, respectively. There are no outliers.

Article Snippet: Samples were incubated with anti-phospho-STAT5 (p-STAT5) Alexa 488 (Y694, clone 47, BD Biosciences) or isotype IgG 1 k Alexa 488 (clone MOPC-21, BD Biosciences), anti-CD33 PE (clone P67.6, BD Biosciences), anti-CD34 APC (clone 8G12, BD Biosciences), anti-CD45 PerCP (clone 2D1, BD Biosciences) and anti-CD38 PE-Cy7 (clone HB7, BD Biosciences) antibodies.

Techniques:

Journal: Blood Cancer Journal

Article Title: Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia

doi: 10.1038/bcj.2013.56

Figure Lengend Snippet: Comparison of p-STAT5-positive cells (%) induced by 0.1 ng/ml of GM-CSF according to the different cell source. In all, 17 JMML BM samples (left box plot), 5 JMML PB samples (middle left box plot), 12 BM samples and 2 PB samples (middle right and right box plot, respectively) from patients with other diseases mimicking JMML are shown. The discriminating threshold (17.17% as assessed in the training set) is indicated. The bold line inside each box plot indicates the median level, while the upper and lower lines indicate the maximum and minimum observed values, respectively. There are no outliers.

Article Snippet: Samples were incubated with anti-phospho-STAT5 (p-STAT5) Alexa 488 (Y694, clone 47, BD Biosciences) or isotype IgG 1 k Alexa 488 (clone MOPC-21, BD Biosciences), anti-CD33 PE (clone P67.6, BD Biosciences), anti-CD34 APC (clone 8G12, BD Biosciences), anti-CD45 PerCP (clone 2D1, BD Biosciences) and anti-CD38 PE-Cy7 (clone HB7, BD Biosciences) antibodies.

Techniques:

Journal: eLife

Article Title: Unsupervised machine learning reveals risk stratifying glioblastoma tumor cells

doi: 10.7554/eLife.56879

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-p-STAT5 (Y694) (mouse-monoclonal) , Fluidigm , RRID: AB_2744690 Cat#3150005A Clone:47 , MC (1:100).

Techniques: Isolation, Software