Journal: Cancers

Article Title: Growth Hormone Upregulates Mediators of Melanoma Drug Efflux and Epithelial-to-Mesenchymal Transition In Vitro and In Vivo

doi: 10.3390/cancers12123640

Figure Lengend Snippet: Subcutaneous B16-F10 mouse melanoma growth in bGH and GHRKO syngeneic mice. ( A ) Subcutaneous B16-F10 tumor growth in male bGH (n = 5) vs. WT mice (n = 6). ( B ) Subcutaneous B16-F10 tumor growth in female bGH (n = 7) and WT mice (n = 6). ( C ) Subcutaneous B16-F10 tumor growth in male GHRKO (n = 6) vs. WT (n = 8) mice. ( D ) Subcutaneous B16-F10 tumor growth in female GHRKO (n = 4) and WT mice (n = 5). Tumor sizes were analyzed by repeated measures (SPSS). ( E ) Weight of subcutaneous B16-F10 tumors from bGH vs. WT mice at dissection. ( F ) Weight of subcutaneous B16-F10 tumors from GHRKO vs. WT mice at dissection. ( G ) GH levels were measured in protein lysates isolated from tumors of bGH and WT mice using ELISA and normalized to total protein concentrations (n = 4). ( H ) Similar GH measurements were performed in protein lysates isolated from tumors of GHRKO and WT mice (males n = 3, females n = 4). ( I ) IGF-1 levels were measured in protein lysates isolated from tumors of bGH and WT mice using ELISA and normalized to total protein concentrations (n = 4). ( J ) Similar IGF-1 measurements were performed in protein lysates isolated from tumors of GHRKO and WT mice (males n = 3, females n = 4). ( K ) Representative images of western blot analysis of phosphorylation (p) and total (t) levels of STAT1, STAT3 and STAT5 in protein lysates isolated from tumors of bGH and WT mice. Densitometry analysis was performed and normalized against β-Actin. The relative expression levels (fold change relative to WT) are labeled under each band. The ratio of phosphorylated vs. total protein levels in tumors from bGH and WT mice are presented in bar graphs (n = 4). ( L ) Representative images of western blot analysis of phosphorylation and total levels of STAT1, STAT3, and STAT5 in protein lysates isolated from tumors of GHRKO and WT mice. (males n = 3, females n = 4). Data are presented as mean ± standard errors (*, p < 0.05, unpaired student’s t -test).

Article Snippet: Efficiency of GHR RNA knockdown was verified by real-time RT-qPCR and confirmed via western blotting for GH-induced tyrosine phosphorylated STAT5 (CST #9351).

Techniques: Dissection, Isolation, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Labeling

Journal: Journal of Cancer

Article Title: CSF2 Overexpression Is Associated with STAT5 Phosphorylation and Poor Prognosis in Patients with Urothelial Carcinoma

doi: 10.7150/jca.14281

Figure Lengend Snippet: Summary of two significantly and differentially expressed genes related to positive regulation of tyrosine phosphorylation of pSTAT5 in the published transcriptome of UBUC (GSE32894).

Article Snippet: The endogenous peroxidase was quenched by saline for 15 minutes and then incubated with primary monoclonal antibodies against CSF2 (1:100, Cat. No. ab77768, rabbit polyclonal, abcam, Cambridge, MA) and phosphor-STAT5 (pSTAT5, Tyr 694/Tyr 699) (1:50, Cat. No. sc-11761, goat polyclonal, Santa Cruz, CA) for an hour.

Techniques: Phospho-proteomics, Cell Differentiation, Expressing, Activity Assay, Binding Assay, Transduction, Migration, Protein Binding

Journal: PLoS ONE

Article Title: Interleukin-15 Plays a Central Role in Human Kidney Physiology and Cancer through the γc Signaling Pathway

doi: 10.1371/journal.pone.0031624

Figure Lengend Snippet: A) Scatchard's plot analysis: Effects of anti-IL-15Rβ and γc mAbs on IL-15 binding to RCC. For the IL-15 binding experiments, RCC7 cells were incubated with increasing concentrations of radioiodinated rIL-15 in presence or not of the following neutralizing mAbs: anti-IL-2Rβ and IL-2Rγ. The nonspecific cell binding was determined in the presence of radioiodinated rhIL-15 and a 100-fold excess of unlabeled rhIL-15. Cell-bound (B) and unbound (free, F) fractions were measured, and the specific bound fraction was calculated by subtracting the nonspecific binding from the cell-bound fraction. On the ordinate is plotted the ratio of the specific bound fraction (expressed in sites per cell) over the total concentration (bound plus free) of radioiodinated rIL-15 (expressed in pM). On the abscissa bound fraction (expressed in sites per cell). The high affinity specific IL-15 binding (Kd = 375 pM, 413 IL-15 binding sites per cell), which was completely abrogated by neutralizing antibody against the IL-2Rβ (inset) but not the γc chain, suggested the presence on RCC of an IL-15Rα/IL-2Rβ complex. B ) Detection of IL-15Rαβ complex by immunoprecipitation (IP) with anti-IL-15Rα (M161) or mouse IgG protein G-Sepharose-conjugate on total lysate (TL) of RCC7. Immunoprecipitated complexes were blotted either with anti-IL-2Rβ (sc-1046) and anti-IL-15Rα (sc-9172). C ) Stimulation for 10 and 40 min with physiologic (10 pg/mL) and supra-physiologic (10 ng/mL) concentrations of rhIL-15 induces the phosphorylation of MAPK ERK1/2 and IκBα in RPTEC and RCC7, whereas STAT5 activation was only observed in RPTEC. Histograms represent densitometry comparison of each factor normalized to β-actin in 3 different RCC (RCC5, RCC7, RCC8) and 3 RPTEC batches. * P<0.05 versus control, Mann-Whitney test. One experiment representative of a total of three is shown.

Article Snippet: Antibodies against phosphorylated ERK (4377), phosphorylated IκB (4921), STAT5 (9358) phosphorylated STAT5 (9356), and the Alexa fluor-conjugated rabbit monoclonal antibody against phosphorylated STAT5 (3939) were obtained from Cell Signaling (Beverly, MA).

Techniques: Binding Assay, Incubation, Concentration Assay, Immunoprecipitation, Phospho-proteomics, Activation Assay, Comparison, Control, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Interleukin-15 Plays a Central Role in Human Kidney Physiology and Cancer through the γc Signaling Pathway

doi: 10.1371/journal.pone.0031624

Figure Lengend Snippet: The γc neutralization, as well as JAK3 or STAT5 inhibition, hamper the maintenance of E-cadherin surface expression induced by rhIL-15 on RPTEC without interfering on the E-cadherin down-regulation on rhIL-15-treated RCC7. Cells were pretreated with 1 µg/ml of neutralizing anti-IL2Rγ antibody mAb2842, 0.25 µM of JAK3 inhibitor (CP-690, 550, Calbiochem) or 100 µM of STAT5 inhibitor (STAT5 Inh., 573108, Calbiochem) for 1 h before adding the recombinant cytokine (10 pg/mL) for 5 days. Treatment with rhIL-15 and STAT5 inhibitor was renewed at day 3. White histograms refer to isotype-matched control. Mean fluorescence intensity values for each marker are shown in each histogram. The data are representative of 3 separate experiments performed using different RCC (RCC5, RCC8) and RPTEC batches.

Article Snippet: Antibodies against phosphorylated ERK (4377), phosphorylated IκB (4921), STAT5 (9358) phosphorylated STAT5 (9356), and the Alexa fluor-conjugated rabbit monoclonal antibody against phosphorylated STAT5 (3939) were obtained from Cell Signaling (Beverly, MA).

Techniques: Neutralization, Inhibition, Expressing, Recombinant, Control, Fluorescence, Marker

Journal: PLoS ONE

Article Title: Interleukin-15 Plays a Central Role in Human Kidney Physiology and Cancer through the γc Signaling Pathway

doi: 10.1371/journal.pone.0031624

Figure Lengend Snippet: RCC7 were transiently transfected for 48 hours with vectors containing IL-2Rγ and/or JAK3 Human cDNA. A ) Transient expression of IL-2Rγ and JAK3 was analyzed by immunoblotting in each transfected RCC. Immunoblotting for β-actin was used as a control for equal protein loading and transfer. B ) Flow cytometry shows that 40 min rhIL-15 treatment did not induce STAT5 phosphorylation in IL-2Rγ- or JAK3-transfected RCC while rhIL-15 treatment induced STAT5 phosphorylation in co-transfected cells. C ) After 48 h, transfected RCC were treated for an additional 48 h with 10 pg/mL of rhIL-15 before evaluating E-cadherin expression by flow cytometry. The introduction of either IL-2Rγ chain, JAK3 or both molecules do not modify E-cadherin expression on untreated rhIL-15 cells, while the E-cadherin down-regulation observed after 48 hours of rhIL-15 treatment was counterbalanced only in co-transfected cells. Mean fluorescence intensity values for each marker are shown in each histogram. One experiment representative of a total of three is shown.

Article Snippet: Antibodies against phosphorylated ERK (4377), phosphorylated IκB (4921), STAT5 (9358) phosphorylated STAT5 (9356), and the Alexa fluor-conjugated rabbit monoclonal antibody against phosphorylated STAT5 (3939) were obtained from Cell Signaling (Beverly, MA).

Techniques: Transfection, Expressing, Western Blot, Control, Flow Cytometry, Phospho-proteomics, Fluorescence, Marker

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Long non-coding RNA RP11-468E2.5 curtails colorectal cancer cell proliferation and stimulates apoptosis via the JAK/STAT signaling pathway by targeting STAT5 and STAT6

doi: 10.1186/s13046-019-1428-0

Figure Lengend Snippet: RP11-468E2.5 and the JAK/STAT signaling pathway-related genes are negatively correlated in CRC tissues. A, The expression of RP11-468E2.5, STAT5, STAT6 and CCND1 detected by RT-qPCR. B, The correlation between RP11-468E2.5 with STAT5, STAT6 and CCND1 using Pearson correlation analysis. JAK, janus kinase; lncRNA, long non-coding RNA; RT-qPCR, reverse transcription quantitative polymerase chain reaction; STAT5, signal transducer and activator of transcription-5; STAT6, signal transducer and activator of transcription-6; CCND1, Cyclin D1; CRC, colorectal cancer. * p < 0.05, compared with the adjacent normal tissues

Article Snippet: The sections were then incubated with primary rabbit polyclonal antibodies against phosphorylated (p)-STAT5 (Tyr694/699, SC-81524, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:100, p-STAT6 (Tyr64, SC-136019, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:200, and Cyclin D1 (CCND1) at 4 °C overnight.

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Long non-coding RNA RP11-468E2.5 curtails colorectal cancer cell proliferation and stimulates apoptosis via the JAK/STAT signaling pathway by targeting STAT5 and STAT6

doi: 10.1186/s13046-019-1428-0

Figure Lengend Snippet: Increased positive expression of p-STAT5, p-STAT6 and CCND1 proteins is evident in CRC tissues. a The expression of p-STAT5, p-STAT6 and CCND1 in CRC tissue detected by immunohistochemistry (× 400). b Antibody labeling conditions following p-STAT5 and p-STAT6, with blocking peptide treatment in immunohistochemistry. c Quantitative analysis for positive expression of p-STAT5, p-STAT6 and CCND1. p-STAT5, p-signal transducer and activator of transcription-5; p-STAT6, p-signal transducer and activator of transcription-6; CRC, colorectal cancer; NC, negative control

Article Snippet: The sections were then incubated with primary rabbit polyclonal antibodies against phosphorylated (p)-STAT5 (Tyr694/699, SC-81524, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:100, p-STAT6 (Tyr64, SC-136019, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:200, and Cyclin D1 (CCND1) at 4 °C overnight.

Techniques: Expressing, Immunohistochemistry, Antibody Labeling, Blocking Assay, Negative Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Long non-coding RNA RP11-468E2.5 curtails colorectal cancer cell proliferation and stimulates apoptosis via the JAK/STAT signaling pathway by targeting STAT5 and STAT6

doi: 10.1186/s13046-019-1428-0

Figure Lengend Snippet: Increased protein expression of STAT5 and STAT6 is found in LOVO, SW620, SW480 and HCT116 cell lines. A and B, Western blot analysis of STAT5, p-STAT5 STAT6, p-STAT6 and GAPDH proteins in different cell lines. C, Subcellular localization of p-STAT5/6 in CRC cells detected by immunofluorescence assay. p-STAT6, p-signal transducer and activator of transcription-6; STAT6, signal transducer and activator of transcription-6; p-STAT5, p-signal transducer and activator of transcription-5; STAT5, signal transducer and activator of transcription-5; GAPDH: glyceraldehyde-3-phosphate dehydrogenase. * p < 0.05, compared with RKO cell line; # p < 0.05, compared with LOVO cell line

Article Snippet: The sections were then incubated with primary rabbit polyclonal antibodies against phosphorylated (p)-STAT5 (Tyr694/699, SC-81524, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:100, p-STAT6 (Tyr64, SC-136019, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:200, and Cyclin D1 (CCND1) at 4 °C overnight.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Long non-coding RNA RP11-468E2.5 curtails colorectal cancer cell proliferation and stimulates apoptosis via the JAK/STAT signaling pathway by targeting STAT5 and STAT6

doi: 10.1186/s13046-019-1428-0

Figure Lengend Snippet: Effect of RP11-468E2.5 on the mRNA expression of JAK/STAT signaling pathway- and apoptosis-related genes in HCT116 and SW480 cells. a RP11-468E2.5 expression and mRNA expression of the JAK/STAT signaling pathway-related genes and apoptosis-related genes determined by RT-qPCR in HCT116 cells. b RP11-468E2.5 expression and mRNA expression of the JAK/STAT signaling pathway- and apoptosis-related genes determined by RT-qPCR in SW480 cells; RT-qPCR, reverse transcription quantitative polymerase chain reaction; NC, negative control; JAK1, janus kinase 1; STAT3, signal transducer and activator of transcription-3; STAT5, signal transducer and activator of transcription-5; STAT6, signal transducer and activator of transcription-6; Bcl-2, B-cell leukemia/lymphoma 2; CCND1, Cyclin D1. * p < 0.05, compared with the blank group

Article Snippet: The sections were then incubated with primary rabbit polyclonal antibodies against phosphorylated (p)-STAT5 (Tyr694/699, SC-81524, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:100, p-STAT6 (Tyr64, SC-136019, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:200, and Cyclin D1 (CCND1) at 4 °C overnight.

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Long non-coding RNA RP11-468E2.5 curtails colorectal cancer cell proliferation and stimulates apoptosis via the JAK/STAT signaling pathway by targeting STAT5 and STAT6

doi: 10.1186/s13046-019-1428-0

Figure Lengend Snippet: Effects of RP11-468E2.5 on the protein expression of JAK/STAT signaling pathway- and apoptosis-related genes in HCT116 and SW480 cells. a and b Western blot analysis of the JAK/STAT signaling pathway- and apoptosis-related proteins in HCT116 cells. c and d Western blot analysis of the JAK/STAT signaling pathway- and apoptosis-related proteins in SW480 cells. NC, negative control; JAK1, janus kinase 1; STAT3, signal transducer and activator of transcription-3; STAT5, signal transducer and activator of transcription-5; STAT6, signal transducer and activator of transcription-6; Bcl-2, B-cell leukemia/lymphoma 2. * p < 0.05, compared with the blank group

Article Snippet: The sections were then incubated with primary rabbit polyclonal antibodies against phosphorylated (p)-STAT5 (Tyr694/699, SC-81524, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:100, p-STAT6 (Tyr64, SC-136019, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:200, and Cyclin D1 (CCND1) at 4 °C overnight.

Techniques: Expressing, Western Blot, Negative Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Long non-coding RNA RP11-468E2.5 curtails colorectal cancer cell proliferation and stimulates apoptosis via the JAK/STAT signaling pathway by targeting STAT5 and STAT6

doi: 10.1186/s13046-019-1428-0

Figure Lengend Snippet: RP11-468E2.5 interacts with STAT5 and STAT6. a Interaction among RP11-468E2.5, STAT5 and STAT6 detected by RNA pull-down assay. b Interaction among RP11-468E2.5, STAT5 and STAT6 verified using RIP assay. STAT5, signal transducer and activator of transcription-5; STAT6, signal transducer and activator of transcription-6. * p < 0.05, compared with IgG group

Article Snippet: The sections were then incubated with primary rabbit polyclonal antibodies against phosphorylated (p)-STAT5 (Tyr694/699, SC-81524, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:100, p-STAT6 (Tyr64, SC-136019, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at a dilution of 1:200, and Cyclin D1 (CCND1) at 4 °C overnight.

Techniques: Pull Down Assay

Journal: Oncotarget

Article Title: Oncogenic STAT5 signaling promotes oxidative stress in chronic myeloid leukemia cells by repressing antioxidant defenses

doi: 10.18632/oncotarget.11480

Figure Lengend Snippet: A . KU812, K562 cell lines were transfected with a dominant negative Flag-STAT5AΔ749-ΔCD4 (Δ5A) bicistronic construct or empty vector (Co). Cells were next incubated with H2DCFDA (5μM) and APC-conjugated anti-CD4 antibody to determine ROS levels in CD4 + transfected cells. (n=3, data are mean ± SEM, * p <0.05). B . Extracts from transfected KU812 and K562 cells were prepared and analyzed by Western blotting with an anti-Flag antibody to verify expression of the dominant negative STAT5AΔ749 mutant (Δ5A). Actin served as a loading control (α-actin). C . KU812 and K562 cells were transfected with shST5/GFP or control shLuc/GFP vectors. Cell lysates were prepared 3 days after transfection and analyzed by Western blotting with indicated antibodies (n=3). D . Quantification of Western blot (ImageJ software) was performed to determine the relative expression of STAT5 (ratio STAT5/actin) in cells transfected with shST5 or shLuc expression vectors (n=3). E . KU812 and K562 cells transfected with shST5/GFP or control shLuc/GFP vectors were stained with CellROX at 3 days post-transfection to quantify ROS levels in GFP + cells (n=5, data are mean ± SEM, *p<0.05; **p<0.01).

Article Snippet: Blots were incubated with the following antibodies (Abs): P-Y 694/699 -STAT5, catalase (Cell Signaling Technology, Danvers, United States), STAT5 (BD Transduction Laboratories, Franklin Lakes, United States), actin (Santa Cruz, Dallas, United States), Flag M2 (Stratagene, Santa Clara, United States), Glutaredoxin1 (Glrx1) (R&D, Minneapolis, United States).

Techniques: Transfection, Dominant Negative Mutation, Construct, Plasmid Preparation, Incubation, Western Blot, Expressing, Mutagenesis, Control, Software, Staining

Journal: Oncotarget

Article Title: Oncogenic STAT5 signaling promotes oxidative stress in chronic myeloid leukemia cells by repressing antioxidant defenses

doi: 10.18632/oncotarget.11480

Figure Lengend Snippet: A . KU812 (10 5 cells/ml) cells were cultured alone in medium (Co) or in HS-27A conditioned medium (+CM HS-27A) or on HS-27A cell monolayers for 72 h (+HS-27A). Cells were stained with 7-AAD and an Alexa Fluor H488-conjugated anti-Ki67 antibody. Concomitant staining with an APC-conjugated anti-CD45 antibody was performed to distinguish leukemic and stromal cells. Cell cycle phase distributions were estimated by flow cytometry. The histogram presents the percentage of leukemic cells (CD45 + cells) in the sub-G1 (apoptotic fraction) and cell cycle phases (n=3, data are mean ± SEM. *p<0.05). B . Cells were stained with the ROS sensitive dye H2DCFDA (5 μM) and an APC-conjugated anti CD45 antibody. ROS levels were then determined in leukemic cells (CD45 + cells) by FACS analysis (n=3 data are mean ± SEM. * p <0.05). C . KU812 cells cultured alone without (Co) or with HS-27A conditioned medium (+CM HS-27A) or on HS-27A cell monolayers (+HS-27A) were isolated using an immunomagnetic CD45 selection kit. KU812 cell extracts were then prepared and subjected to Western blot analysis with indicated antibodies. (n=3). D . Hypothetical model for the role of STAT5 as an inducer of ROS production in CML cells. The pro-oxidant activity of STAT5 is regulated by tyrosine phosphorylation. Constitutive activation of STAT5 (P-Y-STAT5) promotes oxidative stress by repressing expression of catalase and Glrx1. Dephosphorylation of STAT5 might allow the re-expression of catalase and Glrx1 and the decrease of ROS levels in leukemic cells.

Article Snippet: Blots were incubated with the following antibodies (Abs): P-Y 694/699 -STAT5, catalase (Cell Signaling Technology, Danvers, United States), STAT5 (BD Transduction Laboratories, Franklin Lakes, United States), actin (Santa Cruz, Dallas, United States), Flag M2 (Stratagene, Santa Clara, United States), Glutaredoxin1 (Glrx1) (R&D, Minneapolis, United States).

Techniques: Cell Culture, Staining, Flow Cytometry, Isolation, Selection, Western Blot, Activity Assay, Phospho-proteomics, Activation Assay, Expressing, De-Phosphorylation Assay