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: Nucleic Acids Research

Article Title: Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration

doi: 10.1093/nar/gks1310

Figure Lengend Snippet: Summary of genome-wide STAT5 binding sites at L1. (A) The Venn diagram shows the number of identified STAT5A and STAT5B sites (peaks) in AABB tissue and STAT5B sites in BB tissue. (B) Average peak heights of STAT5A and STAT5B in AABB and BB tissues were estimated after library size normalization (RPM, reads per 10 million, input subtracted). (C) Mean fold changes of STAT5A and STAT5B target genes in AABB tissue and STAT5B target genes in BB tissue were calculated. The genes containing STAT5 peaks within ±1 kb around TSSs were regarded as STAT5 target genes. (D) Normalized tag counts (RPM) of STAT5A, RNA polII and H3K4me3 from 200 bp around STAT5A peak centers at L1 were calculated and compared between AABB and BB . Log 2 -transformed values were used ( x and y axes). (E) Normalized tags of H3K4me3 at positions 1 kb upstream and 2 kb downstream of TSS were summed up and divided by the size (3 kb) and then quantile normalized for comparison (top). The scatter plot shows the fold change ( x -axis) and difference ( y -axis) of H3K4me3 average enrichment between genes (spot) in AABB and BB . Cutoffs for significant changes were set as follows: 1.5-fold change ( x -axis, AABB/BB ) and four average tag difference ( y -axis, AABB/BB ). Among the genes showing significant changes of H3K4me3 enrichment, the number of STAT5 target and non-target genes was counted (bottom). (F) Genome browser views represent three gene loci ( Wap , Csn1s2a and Stap1 ) showing changes of H3K4me3 level and one housekeeping gene locus ( Actb ).

Article Snippet: Antibodies against STAT5A (# sc-1081, Santa Cruz, CA, USA), STAT5B (# sc-835, Santa Cruz), RNA polymerase II (# ab5408, Abcam), and histone H3K4me3 (# 17-614, Millipore, Temecula, CA, USA) were used for ChIP.

Techniques: Genome Wide, Binding Assay, Transformation Assay, Comparison

Journal: Nucleic Acids Research

Article Title: Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration

doi: 10.1093/nar/gks1310

Figure Lengend Snippet: Histology and IF staining for NKCC1 of mammary tissues of mice with various STAT5 dosages in early pregnancy. ( A ) Transplanted tissues from mice expressing Stat5a or Stat5b at various levels as indicated were harvested on day 6 of pregnancy and stained with H&E. At this stage alveolar development in all samples is sparse in all epithelial cells expressing Stat5 and is even more reduced in Null cells (f). Black arrows indicate stromal adipocytes and white arrows indicate alveolar epithelial cells. Scale bar = 80 µm. ( B ) Staining of the membrane transporter molecule NKCC1, which is downregulated as epithelial cells differentiate, indicates a more mature developmental stage in wild type (a) cells, intermediate maturity in cells with two or one Stat5 alleles (b and c) and strong staining in Null cells (f). Arrowheads indicate NKCC1-positive cells stained in red. Myoepithelial cells are visualized with antibodies against smooth muscle actin (green).

Article Snippet: Antibodies against STAT5A (# sc-1081, Santa Cruz, CA, USA), STAT5B (# sc-835, Santa Cruz), RNA polymerase II (# ab5408, Abcam), and histone H3K4me3 (# 17-614, Millipore, Temecula, CA, USA) were used for ChIP.

Techniques: Staining, Expressing, Membrane

Journal: Nucleic Acids Research

Article Title: Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration

doi: 10.1093/nar/gks1310

Figure Lengend Snippet: Histology and IF staining for NKCC1 of mammary tissues of mice with various STAT5 dosages at parturition. The nomenclature of mice with the different genotypes is based on the alleles they have retained. We refer to wild-type mice and Stat5ab fl/fl mice as AABB mice; Stat5ab fl/fl;MMTV- Cre (with Stat5ab -deficient mammary epithelial cells) as Null mice; Stat5a −/− mice as BB mice; Stat5b −/− mice as AA mice; Stat5ab +/ null mice as AB mice. Mice carrying only a single functional allele of either Stat5a ( Stat5ab null /Stat5b − ) or Stat5b ( Stat5ab null /Stat5a − ) are referred to as A mice and B mice, respectively. ( A ) Transplanted mammary tissues obtained from mice of different genotypes were collected on the day of parturition and analyzed by histology. Alveoli are expanded and filled with milk in the presence of four (a) and two (b and c) Stat5 alleles. Epithelial cells with only one active Stat5 allele (d and e) form dense alveoli lacking signs of secretory activity. Black arrows indicate stromal adipocytes and white arrows indicate alveolar epithelial cells. Scale bar = 80 µm. ( B ) Mammary tissues of transplanted epithelia obtained from mice of different genotypes were collected at parturition and sections were stained with anti-NKCC1 antibody (red) and α-smooth muscle actin (green). Arrowheads indicate NKCC1-positive cells stained in red. Myoepithelial cells are visualized with antibodies against smooth muscle actin (green).

Article Snippet: Antibodies against STAT5A (# sc-1081, Santa Cruz, CA, USA), STAT5B (# sc-835, Santa Cruz), RNA polymerase II (# ab5408, Abcam), and histone H3K4me3 (# 17-614, Millipore, Temecula, CA, USA) were used for ChIP.

Techniques: Staining, Functional Assay, Activity Assay

Journal: Nucleic Acids Research

Article Title: Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration

doi: 10.1093/nar/gks1310

Figure Lengend Snippet: STAT5 regulation of and binding to known target genes

Article Snippet: Antibodies against STAT5A (# sc-1081, Santa Cruz, CA, USA), STAT5B (# sc-835, Santa Cruz), RNA polymerase II (# ab5408, Abcam), and histone H3K4me3 (# 17-614, Millipore, Temecula, CA, USA) were used for ChIP.

Techniques: Binding Assay, Expressing

Journal: Nucleic Acids Research

Article Title: Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration

doi: 10.1093/nar/gks1310

Figure Lengend Snippet: STAT5 binding and chromatin features of the casein gene cluster. Genome browser tracks represent enrichment of STAT5A, STAT5B, H3K4me3 and RNA PolII in wild type ( AABB ) and Stat5a-null mammary tissues as well as STAT5 in liver and T cells. The liver and T-cell STAT5 ChIP-seq data sets were obtained from previous studies (GSE31578 and GSE36890). Conservation of GAS motifs (TTCnnnGAA) was calculated using the GERP score (the higher score means higher conservation) . Expression level of five milk protein genes was measured by both RNA-seq and qRT-PCR (bottom left). Absolute expression level of the milk protein genes is shown (bottom right).

Article Snippet: Antibodies against STAT5A (# sc-1081, Santa Cruz, CA, USA), STAT5B (# sc-835, Santa Cruz), RNA polymerase II (# ab5408, Abcam), and histone H3K4me3 (# 17-614, Millipore, Temecula, CA, USA) were used for ChIP.

Techniques: Binding Assay, ChIP-sequencing, Expressing, RNA Sequencing, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Sequential activation of genetic programs in mouse mammary epithelium during pregnancy depends on STAT5A/B concentration

doi: 10.1093/nar/gks1310

Figure Lengend Snippet: STAT5 binding and chromatin features of STAT5 target genes. Genome browser tracks represent enrichment of STAT5A, STAT5B, H3K4me3 and RNA PolII in wild type ( AABB ) and Stat5a-null mammary tissues as well as STAT5 in liver and T cells. The liver and T-cell STAT5 ChIP-seq data sets were obtained from previous studies (GSE31578 and GSE36890). Conservation of GAS motifs (TTCnnnGAA) was calculated using the GERP score (the higher score means more conserved). ( A ) Wap gene, ( B ) Cish gene, ( C ) Socs2 gene, ( D ) Bcl6 gene and ( E ) Stat5a/b genes.

Article Snippet: Antibodies against STAT5A (# sc-1081, Santa Cruz, CA, USA), STAT5B (# sc-835, Santa Cruz), RNA polymerase II (# ab5408, Abcam), and histone H3K4me3 (# 17-614, Millipore, Temecula, CA, USA) were used for ChIP.

Techniques: Binding Assay, ChIP-sequencing

Journal: American Journal of Cancer Research

Article Title: MiRNA-1469 promotes lung cancer cells apoptosis through targeting STAT5a

doi:

Figure Lengend Snippet: Stat5a is a direct target of miRNA-1469. A. MiR-1469 targeting site resides at nucleotides 226-232 of Stat5a-3’UTR and is highly conserved in different species. Upper panel: sequence alignment of miR-1469 with binding sites on the Stat5a-3’UTR. Bottom panel: sequence of the miR-1469 binding site within the Stat5a-3’UTR of three species (human, chimpanzee and rhesus). B. Diagram of the luciferase reporter plasmids including plasmid with the full-length Stat5a-3’UTR insert (pIS0-Stat5a-3’UTR) and plasmid with a mutant Stat5a-3’UTR (pIS0-Stat5a-3’UTR-mut) which carried a substitution of three nucleotides within the miR-1469 binding site. C. Luciferase activity assay demonstrates a direct targeting of the Stat5a-3’UTR by miR-1469. A549 cells (left panel) and H1650 cells (right panel) were transfected with miR-1469 mimics (20 nM) and pIS0-Stat5a-3’UTR /pIS0-Stat5a-3’UTR-mut. pRL-SV40 Renilla was used for the normalization of transfection efficiency. After 48 h, the luciferase activities were measured. D. Western blotting was used to detect the expression of the Stat5a protein after miRNA-1469 mimics (20 nM) or miRNA-1469; Inhibitor (40 nM) transfection of A549 (left panel) or H1650 (right panel) cells. E. Stat5a mRNA in the A549 (left panel) or H1650 (right panel) cell lines treated as above was measured with real-time RT-PCR. β-actin was used as internal control. F. Overexpression of miR-1469 mimics (20 nM) or miRNA-1469.Inhibitor (40 nM) in A549 or H1650 cell lines transfected miRNA-1469 mimics or miRNA-1469. Inhibitor was measured by real-time RT-PCR. U6 was used as internal control. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The antibodies against Stat5a (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-1081), phosphorylated H2AX (gH2AX) (Cell Signaling Technology, Beverly, MA, USA, # 9718), Bcl-2 and b-actin (Cell Signaling Technology, #4970) were respectively used to detect their targeting proteins: Statistical analysis Data were presented as mean±SD from at least three separate experiments, and Student’s t-test analysis was performed using SPSS 17.0 software.

Techniques: Sequencing, Binding Assay, Luciferase, Plasmid Preparation, Mutagenesis, Activity Assay, Transfection, Western Blot, Expressing, Quantitative RT-PCR, Control, Over Expression

Journal: American Journal of Cancer Research

Article Title: MiRNA-1469 promotes lung cancer cells apoptosis through targeting STAT5a

doi:

Figure Lengend Snippet: MiR-1469 regulates apoptosis through Stat5a. A. Apoptosis of A549 cell were detected by FCM 48 h after miRNA-1469 mimics and combined with Stat5a transfection (left panel). Error bars denote mean ± SD (right panel). B. The apoptosis of H1650 cells 48 h after transfection of miRNA-1469 mimics and combined with Stat5a were detected by FCM (left panel). Error bars denote mean ± SD (right panel). **, p < 0.01; ***, p < 0.001.

Article Snippet: The antibodies against Stat5a (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-1081), phosphorylated H2AX (gH2AX) (Cell Signaling Technology, Beverly, MA, USA, # 9718), Bcl-2 and b-actin (Cell Signaling Technology, #4970) were respectively used to detect their targeting proteins: Statistical analysis Data were presented as mean±SD from at least three separate experiments, and Student’s t-test analysis was performed using SPSS 17.0 software.

Techniques: Transfection