Journal: American Journal of Cancer Research

Article Title: DOT1L promotes cell proliferation and invasion by epigenetically regulating STAT5B in renal cell carcinoma

doi:

Figure Lengend Snippet: DOT1L silencing demethylates H3K79 and downregulates the transcription of STAT5B. A. A Venn diagram was utilized to identify coexpressed DEGs. B. GSEA showed that DOT1L was closely linked to the KEGG pathway JAK-STAT SIGNALING. C. Pearson correlation between DOT1L and STAT5B expression in KIRC from GEPIA. D. The mRNA level of STAT5B was measured by qPCR in DOT1L knockdown 786-O cells. E. The DOT1L, H3K79me2, STAT5B and p-STAT5 protein levels were measured by Western blotting in DOT1L knockdown 786-O cells. F. ChIP-seq of H3K79me2 in the ENCODE database showing occupancy on STAT5B promoter in different human cancer cell lines. G. ChIP primer region for human STAT5B. H, I. The ChIP assay showed H3K79me2 occupancy on the STAT5B promoter in 786-O cells by agarose gel electrophoresis and qPCR.

Article Snippet: Antibodies against cyclin A2 (CCNA2; #91500), Bcl2-associated X protein (Bax; #5023), and phospho-STAT5 (Tyr694) (#4322) were purchased from Cell Signaling Technology (Massachusetts, USA).

Techniques: Expressing, Knockdown, Western Blot, ChIP-sequencing, Agarose Gel Electrophoresis

Journal: American Journal of Cancer Research

Article Title: DOT1L promotes cell proliferation and invasion by epigenetically regulating STAT5B in renal cell carcinoma

doi:

Figure Lengend Snippet: Overexpression of STAT5B partially rescues DOT1L silencing-induced cell proliferation inhibition and cell cycle arrest. A. Assessment of the DNA transfection efficiency after incubation with the pEGFP-N1/STAT5B vector (oeSTAT5B) or the pEGFP-N1 (sham) vector in 786-O cells with sh2-mediated DOT1L knockdown at the 24 h time point. B. DOT1L and STAT5B mRNA expression was measured by using qPCR in 786-O cells after DOT1L silencing and STAT5B overexpression. C. DOT1L, STAT5B and p-STAT5 protein levels were measured by Western blotting in 786-O cells after DOT1L knockdown and STAT5B overexpression. D. The cell growth curve was constructed with CCK8 assay data in 786-O cells after DOT1L knockdown and STAT5B overexpression. Both the sh2 and sh2+sham groups were used as the vector controls for STAT5B overexpression at the 0/24/48/72 h time points. E. The cell cycle was analyzed by flow cytometry in 786-O cells with DOT1L knockdown and STAT5B restoration. F. Cyclin A2, P21, and CDK6 protein expression was measured by Western blotting in 786-O cells after DOT1L knockdown and STAT5B overexpression.

Article Snippet: Antibodies against cyclin A2 (CCNA2; #91500), Bcl2-associated X protein (Bax; #5023), and phospho-STAT5 (Tyr694) (#4322) were purchased from Cell Signaling Technology (Massachusetts, USA).

Techniques: Over Expression, Inhibition, Transfection, Incubation, Plasmid Preparation, Knockdown, Expressing, Western Blot, Construct, CCK-8 Assay, Flow Cytometry

Journal:

Article Title: FoxO3a and BCR-ABL Regulate cyclin D2 Transcription through a STAT5/BCL6-Dependent Mechanism

doi: 10.1128/MCB.24.22.10058-10071.2004

Figure Lengend Snippet: Expression of BCL6 and STAT5 in BV173 cells after STI571 treatment and effect of FoxO3a expression on BCL6 promoter activity. (A) BV173 cells were treated for the indicated times with STI571. Cell lysates were prepared at the indicated times, separated on polyacrylamide gels, and subjected to immunoblotting with specific antibodies for BCL6 and STAT5. (B) BV173 cells were treated with STI571 for the times indicated, and total RNA was extracted and analyzed for expression of BCL6. Also shown is the expression of GAPDH as a control for RNA loading and blotting. (C) BV173 cells were transiently transfected with pGL3.1296 or pGL3.1296.mutBCL6 promoter reporter constructs (10 μg) along with pLPC-FoxO3a(wt), pLPC-FoxO3a(A3), or the empty vector (5 μg). Cells were harvested 24 h after transfection and assayed for luciferase activity. Values are corrected for cotransfected Renilla activity and expressed as arbitrary units. The data shown represent the averages of results from two independent experiments, and the error bars show the standard deviation.

Article Snippet: The antibodies against BCL6 (C-19) and cdk4 (C-22) were from Santa Cruz Biotechnology, anti-STAT5 was from Zymed, anti-phospho-STAT5 (Tyr694) was from Cell Signaling, and anti-FoxO3a and anti-phospho-FoxO3a (Thr32) were purchased from Upstate Biotechnology.

Techniques: Expressing, Activity Assay, Western Blot, Transfection, Construct, Plasmid Preparation, Luciferase, Standard Deviation

Journal:

Article Title: FoxO3a and BCR-ABL Regulate cyclin D2 Transcription through a STAT5/BCL6-Dependent Mechanism

doi: 10.1128/MCB.24.22.10058-10071.2004

Figure Lengend Snippet: Electrophoretic mobility shift analysis of STAT5 DNA-binding complexes after STI571 treatment of BV173 cells. (A) DNA-binding protein extracts were prepared from BV173 cells at the indicated times after STI571 treatment. The extracts were then used for gel mobility shift experiments with a 32P-labeled oligonucleotide probe containing the STAT5 binding site in the human cyclin D2 promoter. (B) Antibody supershift analysis of components of STAT5 complexes at different times after STI571 treatment. Supershifts were performed using specific antibodies against STAT5 and BCL6, as indicated, on whole-cell extracts prepared from cells at 0, 1, and 4 h after STI571 treatment.

Article Snippet: The antibodies against BCL6 (C-19) and cdk4 (C-22) were from Santa Cruz Biotechnology, anti-STAT5 was from Zymed, anti-phospho-STAT5 (Tyr694) was from Cell Signaling, and anti-FoxO3a and anti-phospho-FoxO3a (Thr32) were purchased from Upstate Biotechnology.

Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Mobility Shift, Labeling

Journal:

Article Title: FoxO3a and BCR-ABL Regulate cyclin D2 Transcription through a STAT5/BCL6-Dependent Mechanism

doi: 10.1128/MCB.24.22.10058-10071.2004

Figure Lengend Snippet: ChIP assay of BV173 cells after STI571 treatment. (A) BV173 cells were treated with STI571 for 4 h. Protein-DNA complexes were formaldehyde cross-linked in vivo. Chromatin fragments from these cells were subjected to immunoprecipitation with antibodies to STAT5 or BCL6 as indicated. After cross-link reversal, the coimmunoprecipitated DNA was amplified by PCR using the A (−1446/−1149), B (−713/−499), and C (−548/−323) primers and resolved in 2% agarose gel. The PCR products obtained with A primers were also detected by Southern blotting, and shown is the quantitation by phosphorimaging of the results obtained. (B) BV173 cells were treated with STI571 for 4 or 8 h. Protein-DNA complexes were formaldehyde cross-linked in vivo. Chromatin fragments from these cells were subjected to immunoprecipitation with a specific antibody to FoxO3a. After cross-link reversal, the coimmunoprecipitated DNA was amplified by PCR using the A (−1446/−1149), D (−839/−501), and E (+38/+349) primers and resolved in 2% agarose gel.

Article Snippet: The antibodies against BCL6 (C-19) and cdk4 (C-22) were from Santa Cruz Biotechnology, anti-STAT5 was from Zymed, anti-phospho-STAT5 (Tyr694) was from Cell Signaling, and anti-FoxO3a and anti-phospho-FoxO3a (Thr32) were purchased from Upstate Biotechnology.

Techniques: In Vivo, Immunoprecipitation, Amplification, Agarose Gel Electrophoresis, Southern Blot, Quantitation Assay

Journal: Cancers

Article Title: A Novel Inhibitor of STAT5 Signaling Overcomes Chemotherapy Resistance in Myeloid Leukemia Cells

doi: 10.3390/cancers11122043

Figure Lengend Snippet: 17f associated with IM inhibit STAT5 activity in resistant K562R cells. ( A ) K562S or K562R cells transfected with a 6×(STAT5)-TK-luciferase reporter construct or a control TK-luciferase vector were treated or not (Co) with 17f (5 µM), IM (1 µM) or with the combination of 17f and IM for 48 h. Luciferase activities were then determined as described in Methods. Luciferase activity (arbitrary units) in the histogram represents the relative luminescence unit (rlu) values/mg of proteins ( n = 3 in triplicates, data are mean ± SD, * p < 0.05, ** p < 0.001). ( B ) qRT-PCR analysis of PIM1 and CISH expression in K562S and K562R treated or not (Co) with IM (1 µM),17f (5 µM) or with combined 17f and IM for 24 h. Results are presented as the fold change in PIM1 and CISH gene expression in treated cells normalized to internal control genes ( GAPDH , ACTB and RPL13a ) and relative to control condition (normalized to 1) ( n = 3 in triplicates, data are mean ± SD, * p < 0.05; one-sample t -test).

Article Snippet: Blots were incubated with the following antibodies (Abs): P-Y 694/699 -STAT5, Actin (Cell Signaling Technology, Danvers, MA, USA), STAT5 (BD Transduction Laboratories, Franklin Lakes, NJ, USA), STAT5A and STAT5B (Zymed/ThermoFisher Scientific, Waltham, MA, USA).

Techniques: Activity Assay, Transfection, Luciferase, Construct, Control, Plasmid Preparation, Quantitative RT-PCR, Expressing, Gene Expression

Journal: Cancers

Article Title: A Novel Inhibitor of STAT5 Signaling Overcomes Chemotherapy Resistance in Myeloid Leukemia Cells

doi: 10.3390/cancers11122043

Figure Lengend Snippet: 17f associated with IM inhibits STAT5B protein expression in K562R cells ( A ) Protein extracts from K562S and K562R cells treated with 17f 5 µM or DMSO with or without IM for 24 h were analyzed by western blotting to detect P-Y 694/699 -STAT5 and STAT5 protein expression ( n = 2). Actin served as the loading control. ( B ) qRT-PCR analysis of STAT5A and STAT5B expression in K562R cultured with IM (1 µM) as control or treated with 17f (5µM) and IM for 24 h. Results are presented as the fold change in STAT5A and STAT5B gene expression in treated cells normalized to internal control genes ( GAPDH , ACTB and RPL13a ) and relative to control condition (normalized to 1) ( n = 3 in triplicates, data are mean ± SD, * p < 0.05; one sample t -test). ( C ) Expression of STAT5A and STAT5B proteins in K562R cells treated or not with 17f (5 µM) was analyzed by western blot ( n = 2). Actin served as the loading control.

Article Snippet: Blots were incubated with the following antibodies (Abs): P-Y 694/699 -STAT5, Actin (Cell Signaling Technology, Danvers, MA, USA), STAT5 (BD Transduction Laboratories, Franklin Lakes, NJ, USA), STAT5A and STAT5B (Zymed/ThermoFisher Scientific, Waltham, MA, USA).

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR, Cell Culture, Gene Expression

Journal: Cancers

Article Title: A Novel Inhibitor of STAT5 Signaling Overcomes Chemotherapy Resistance in Myeloid Leukemia Cells

doi: 10.3390/cancers11122043

Figure Lengend Snippet: 17f promotes apoptosis, cell cycle arrest and inhibition of STAT5B protein expression in MV4-11R cells. ( A ) Flow cytometry histogram of MV4-11R cells cultured for 48 h with Ara-C and 17f or Ara-C and DMSO as control. Cells were stained with anti-annexin V coupled with FITC and with 7-AAD to determine the percentages of apoptotic cells ( n = 3 in triplicates, data are mean ± SD, ** p < 0.01). ( B ) MV4-11R cells treated for 48 h with Ara-C and 17f or Ara-C and DMSO as control were stained with 7-AAD and an Alexa Fluor 488-conjugated anti-Ki67 antibody. Cell cycle phase distributions were then estimated by flow cytometry. The histogram presents the percentage of cells in the G 0 phase ( n = 3 in triplicates, data are mean ± SD, * p < 0.05, *** p < 0.001). ( C ) Protein extracts from MV4-11R cells treated with Ara-C and 17f or Ara-C and DMSO for 24 h were analyzed by immunoblotting to detect P-Y 694/699 -STAT5 and STAT5 protein expression ( n = 2). Actin served as the loading control. ( D ) Expression of STAT5A and STAT5B proteins in MV4-11R cells treated or not with 17f (5 µM) was also analyzed by western blot ( n = 2).

Article Snippet: Blots were incubated with the following antibodies (Abs): P-Y 694/699 -STAT5, Actin (Cell Signaling Technology, Danvers, MA, USA), STAT5 (BD Transduction Laboratories, Franklin Lakes, NJ, USA), STAT5A and STAT5B (Zymed/ThermoFisher Scientific, Waltham, MA, USA).

Techniques: Inhibition, Expressing, Flow Cytometry, Cell Culture, Control, Staining, Western Blot

Journal: Biology of reproduction

Article Title: CDH1 is a specific marker for undifferentiated spermatogonia in mouse testes.

doi: 10.1095/biolreprod.106.053181

Figure Lengend Snippet: FIG. 1. CDH1 was expressed in a small subset of cells in the seminiferous tubules of mouse testes. a) Immunohistochemical analyses of seminiferous tubules in mature mouse testes showed that CDH1 was expressed in a small fraction of germ cells (arrows). The section was counterstained with hematoxylin. Bar ¼ 20 lm. b) Double staining with CDH1 (green) and GATA4 (red). GATA4 was expressed in the nuclei of Sertoli cells and Leydig cells. CDH1-posi- tive cells (arrows) were located on the basement membrane in seminiferous tu- bules and were negative for GATA4. The strong green fluorescence shown in the cytoplasm of Leydig cells was autofluores- cence. Bar ¼ 20 lm. c) Western blot analysis of CDH1 (arrow, about 124 kDa) from mouse testes. Lysate from testes (lane 1) and the COS7 cells transfected with the CDH1 cDNA expression vector (lane 2) and control COS7 (lane 3) were electropho- resed. Left panel shows the Western blot with the monoclonal antibody (ECCD-2), and right panel shows the Western blot with the polyclonal antibody. The extra band of about 90 kDa shown in lane 3 of the right panel was probably the degradation product of ectopically expressed CDH1. d) Double staining of CDH1 with polyclonal antibody (left), monoclonal antibody (middle), and merged images (right). e) CDH1-positive cells had oval-shaped nuclei displaying no heterochromatin with DAPI staining (mid- dle, arrowhead). f–h) Double staining of CDH1 (left) and ITGA6 (f, middle), ITGB1 (g, middle), or TACSTD1 (h, middle) and merged images (right). CDH1-positive cells also were positive for ITGA6, ITGB1, and TACSTD1. Bar ¼ 10 lm.

Article Snippet: The cryosections were incubated with 2 lg/ml goat anti-mouse CDH1 antibody or 10 lg/ml rat anti-mouse CDH1 and either rat anti-human ITGA6 (MCA 699; Serotec, Kidlington, UK), rat anti-mouse ITGB1 (MAB 1977; Chemicon, Temecula, CA), goat anti-mouse GATA4 (sc-1237; Santa Cruz Biotechnology), rabbit anti-rat CYP11A1 (AB1244; Chemicon), goat anti-human VIMENTIN (sc-7557; Santa Cruz Biotechnology), or rat anti-TACSTD1 (Developmental Studies Hybridoma Bank, Iowa City, IA) antibody.

Techniques: Immunohistochemical staining, Double Staining, Membrane, Fluorescence, Western Blot, Transfection, Expressing, Plasmid Preparation, Control, Staining

Journal: Cell stem cell

Article Title: Smooth muscle cell reprogramming in aortic aneurysms

doi: 10.1016/j.stem.2020.02.013

Figure Lengend Snippet: (A) Representative IMC images stained for GFP (green), CD105 (red), CD73 (blue), CD90 (yellow), Sca-1 (magenta), and CD44 (cyan) in the overlaid format and individual channel images (N=6 mice/group). Scale bar: 500 μm. (B) PhenoGraph clustering of all cell phenotype visualized as a distinct color on the tSNE plot. (C) tSNE plot of high-dimension single cell data. (D) Heatmap visualizing the marker intensity for each Phenograph. (E) (left) Cluster 17 location in the tissue section using HistoCAT spatial clustering analysis. (right) A high-magnification of (Ea) left. (F) A cartoon showing confetti color distribution in bone nodule area and adjacent to the bone nodule area. (G) TGFβR2iSMC-Apoe mice VSMCs adjacent to the bone nodule area are mixed labeled with red fluorescent protein (RFP), yellow fluorescent protein (YFP), nuclear (n) green fluorescent protein (GFP), or membrane associated (m) cyan fluorescent protein (CFP). Scale bar: 10 μm. (H) TGFβR2iSMC-Apoe mice VSMCs in the bone nodule media areas are labeled with single color fluorescent protein after 4 months of high cholesterol high fat diet. Scale bar: 10 μm. (I) Bar chart showing the proportions of each of the Confetti colors in TGFβR2iSMC-Apoe mice VSMCs in media ascending aorta adjacent to the bone nodule area and in bone nodule area. N=10 for TGFβR2iSMC-Apoe mice. See also Figure S4, Figure S5, and Table S2B.

Article Snippet: Mouse monoclonal anti-CD44 , NOVUS BIOLOGICALS , Cat#NBP1-47386, RRID:AB_10010339.

Techniques: Staining, Marker, Labeling, Membrane

Journal: Cell stem cell

Article Title: Smooth muscle cell reprogramming in aortic aneurysms

doi: 10.1016/j.stem.2020.02.013

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-CD44 , NOVUS BIOLOGICALS , Cat#NBP1-47386, RRID:AB_10010339.

Techniques: Recombinant, Multiplex Assay, Staining, In Situ, Marker, Expressing, Plasmid Preparation, shRNA, Software

Journal: Cell reports

Article Title: IL-2 can signal via chemokine receptors to promote regulatory T cells’ suppressive function

doi: 10.1016/j.celrep.2023.112996

Figure Lengend Snippet: (A) Binding of soluble MAdCAM-1 to CD4 T cells treated in the presence or absence of inhibitory anti-IL-2 receptor β (TMB1) with PC61 (5 μg/mL), IL-2 (2.5 μg/mL), or PMA (100 nM). GFP + Tregs were analyzed. Data represent mean ± SEM (n = 6). One-way ANOVA with Bonferroni post-test. NS, not significant; ***p < 0.001. (B) STAT5 phosphorylation of CD4 T cells in the presence of PC61 (5 μg/mL) or IL-2 (2.5 μg/mL). GFP + Tregs were analyzed. Data are representative of at least three independent experiments. (C) STAT5 phosphorylation response of GFP + Tregs to IL-2 in the presence or absence of 100 nM baricitinib. Data represent mean ± SEM (n = 6). (D) Binding of soluble MAdCAM-1 to GFP + Tregs treated with 5 μg/mL control IgG (−) or PC61. The addition of 100 nM baricitinib did not significantly affect the response to PC61. Data represent mean ± SEM (n = 9). One-way ANOVA with Bonferroni post-test. NS, not significant. (E) Binding of soluble MAdCAM-1 to CD4 T cells treated with indicated inhibitors in the presence of PC61 (5 μg/mL) or IgG control. GFP + Tregs were analyzed. Data represent mean ± SEM (n = 6). One-way ANOVA with Bonferroni post-test. (F) Binding of soluble MAdCAM-1 to CD4 T cells treated with pertussis toxin (PTX) (100 nM) or vehicle in the presence of PC61 (5 μg/mL), CXCL12 (1 μg/mL), or PMA (100 nM). GFP + Tregs were analyzed. Data represent mean ± SEM (n = 6). One-way ANOVA with Bonferroni post-test. (G) The key signaling events that occur downstream of GPCR signaling that lead to integrin activation. (H) PTX blocks PC61 enhancement of Tregs’ suppressive activity. Tregs isolated from CD45.2 congenic Foxp3 GFP mice were mixed with responder cells at the indicated Treg/responder cell ratios in the presence of PC61 (5 μg/mL), PTX (100 nM), or PC61 together with PTX. Responder cells were CFSE-labeled CD45.1 congenic C57BL/6 CD4 + CD25 − naive T cells activated by anti-CD3 (5 μg/mL), anti-CD28 (5 μg/mL), and IL-2 (6 ng/mL). CFSE populations gated on CD45.1 + cells were analyzed by flow cytometry on day 5 to determine the division index using FlowJo software. Data represent mean ± SEM (n = 4). Two-way ANOVA with Bonferroni post-test. NS, not significant; **p < 0.01; ***p < 0.001.

Article Snippet: Phospho-STAT5 (Tyr694) (C71E5) Rabbit mAb (PE Conjugate) #5387 , Cell Signaling Technology , 5387;RRID:AB_2797611.

Techniques: Binding Assay, Phospho-proteomics, Control, Activation Assay, Activity Assay, Isolation, Labeling, Flow Cytometry, Software

Journal: Cell reports

Article Title: IL-2 can signal via chemokine receptors to promote regulatory T cells’ suppressive function

doi: 10.1016/j.celrep.2023.112996

Figure Lengend Snippet:

Article Snippet: Phospho-STAT5 (Tyr694) (C71E5) Rabbit mAb (PE Conjugate) #5387 , Cell Signaling Technology , 5387;RRID:AB_2797611.

Techniques: Control, Recombinant, Purification, Produced, Virus, Bacteria, Electron Microscopy, Emulsion, Staining, Cell Culture, Expressing, Transfection, Cell Isolation, Enzyme-linked Immunosorbent Assay, Endotoxin Assay, cDNA Synthesis, Mutagenesis, Plasmid Preparation, Software

Journal: Pharmaceuticals

Article Title: The Inhibition of Stat5 by a Peptide Aptamer Ligand Specific for the DNA Binding Domain Prevents Target Gene Transactivation and the Growth of Breast and Prostate Tumor Cells

doi: 10.3390/ph6080960

Figure Lengend Snippet: Infection with a lentivirus gene transfer vector encoding Stat5-directed shRNA reduces Stat5 expression and suppresses the growth and viability of human tumor cells. ( a ) A431 epidermal and PC-3 prostate carcinoma cell lines were infected with lentiviruses encoding either the empty vector (LeGO-G: eGFP), a scrambled shRNA (LeGO-C: mCherry) or pairs of shRNA specific for the mRNA of the human Stat5a and Stat5b isoforms.6 days after infection knockdown-efficiency was verified by western blot, using antibodies recognizing either total or tyrosine phosphorylated Stat5 protein. The detection of total Stat3 served as a control of shRNA specificity. Eight days after infection cell density and morphology was documented by phase contrast and fluorescence microscopy. ( b ) Over a period of 20 days after viral transduction changes in proliferation and viability of A431 and PC-3 cells were measured by XTT assay at regular intervals. The infection with empty vector and Stat5-shRNA expressing lentiviruses was done in triplicates and the simultaneous infection with scrambled shRNA expressing lentiviruses was done once. Results are shown as the percentage of viable cells compared to the mock-treated control. ( c ) The same experiments were performed with the T-47D breast and HCT116 colorectal cancer cell line. (n = 3; Ø ± SD) Significantly reduced XTT-values in comparison to empty vector expressing cells are indicated. * p < 0.05, ** p < 0.01, *** p < 0.001 (2-way-ANOVA with Bonferroni correction).

Article Snippet: P-STAT5 (C11C5, *9359: Tyr694 Stat5a; Tyr699 Stat5b) and P-STAT5-Alexa Fluor ® 647 (C71E5, *9365: Tyr694 Stat5a; Tyr699 Stat5b) antibodies were purchased from Cell Signaling (Cambridge, UK).

Techniques: Infection, Plasmid Preparation, shRNA, Expressing, Knockdown, Western Blot, Control, Fluorescence, Microscopy, Transduction, XTT Assay, Comparison

Journal: Pharmaceuticals

Article Title: The Inhibition of Stat5 by a Peptide Aptamer Ligand Specific for the DNA Binding Domain Prevents Target Gene Transactivation and the Growth of Breast and Prostate Tumor Cells

doi: 10.3390/ph6080960

Figure Lengend Snippet: Identification of a 12mer peptide aptamer sequence (S5-DBD-PA) that specifically binds with high affinity to the DNA-binding domain (DBD) of Stat5. ( a ) Yeast-two-hybrid (Y2H) screening strategy for the isolation of PA sequences that interact with a functional domain of human Stat5. Y2H screens were performed with a yeast strain (KF1) containing four different Gal4-dependent reporter genes of different stringency. This included genes for the biosynthesis of histidine ( His-3 ), adenine ( Ade-2 ) and uracil ( Ura-3 ) as well as the LacZ -reporter gene for β-galactosidase expression and activity measurement. The corresponding expression vectors (bait and prey) were co-transformed into yeast cells, which were plated and grown under highly stringent conditions on selective media lacking uracil, for identifying the strongest PA interactions. The clone with the best binding properties after revalidation was chosen for further analysis. ( b ) Schematic representation of the cloned bait and prey fusion proteins used for Y2H screening. The DBD of human Stat5A comprises amino acids 331 to 473, indicated in red in the crystallographic structure of a Stat-dimer bound to DNA . This domain was fused to the Gal4 DNA-binding domain (Gal4-DBD) and used as bait. The complex 12-mer PA library was expressed within the active loop of the modified human thioredoxin scaffold (hTRX Δcys5 , schematically indicated by the hTRX crystal structure), which was fused to the Gal4 transcriptional activation domain (Gal4-AD). These random PA sequences presenting fusion proteins were used as prey constructs.( c ) The specificity of binding of the identified aptamer (S5-DBD-PA) to the STAT5-DBD was verified after plasmid sequencing and retransformation by plating the yeast cultures in 1:10, 1:100 and 1:1,000 dilutions on selective media lacking leucine (L) and tryptophan (T), which synthesis genes were encoded by the corresponding bait and prey expression vectors, and additionally either histidine (H), adenine (A) or uracil (U). Weak interactions allow growth on -LTH, whereas growth on -LTU requires strong interactions. The interaction of p53 or lamin C with the SV40 Large T antigen served as positive and negative controls. The Gal4-AD (GAD) fused S5-DBD-PA binding properties against the single GAL4-DBD (GBD) and other fused domains of Stat3 and Stat5 (DBD- or SH2-domain) were additionally evaluated. Protein interactions were also quantified by measuring β-galactosidase activity. Results are shown as Miller-Units (n = 3; Ø ± SD).

Article Snippet: P-STAT5 (C11C5, *9359: Tyr694 Stat5a; Tyr699 Stat5b) and P-STAT5-Alexa Fluor ® 647 (C71E5, *9365: Tyr694 Stat5a; Tyr699 Stat5b) antibodies were purchased from Cell Signaling (Cambridge, UK).

Techniques: Sequencing, Binding Assay, Isolation, Functional Assay, Expressing, Activity Assay, Transformation Assay, Clone Assay, Modification, Activation Assay, Construct, Plasmid Preparation

Journal: Pharmaceuticals

Article Title: The Inhibition of Stat5 by a Peptide Aptamer Ligand Specific for the DNA Binding Domain Prevents Target Gene Transactivation and the Growth of Breast and Prostate Tumor Cells

doi: 10.3390/ph6080960

Figure Lengend Snippet: Domain structure of the S5-DBD-PA protein construct used for viral and protein transduction experiments and a model for its inhibitory function. ( a ) The Stat5-DBD specific 12mer PA sequence (S5-DBD-PA) was introduced in the protruding active loop of the hTRX Δcys5 protein, which serves as a scaffold for enhanced binding affinity and stability. A protein transduction domain (PTD) consisting of 9 arginines (9-R) is added for intracellular uptake. Additionally a C- terminal histidine tag (His) for purification, a NLS-domain for an enhanced nuclear import and a N-terminal Flag-tag (Flag) for proper detection were added. The complete S5-DBD-PA protein construct has a size of 21 kDa. As a negative control for further analysis the scaffold protein, containing an unspecific 9mer linker sequence instead of the PA sequence was used. This control protein was termed hTRX and has a size of 20 kDa. ( b ) Model for inhibition of the Stat5-DNA-binding activity by the interacting peptide ligand S5-DBD-PA. The specific interaction of S5-DBD-PA with the Stat5-DBD prevents the binding of Stat5 and its DNA response element. It blocks essential regions for the recognition and binding to GAS DNA.

Article Snippet: P-STAT5 (C11C5, *9359: Tyr694 Stat5a; Tyr699 Stat5b) and P-STAT5-Alexa Fluor ® 647 (C71E5, *9365: Tyr694 Stat5a; Tyr699 Stat5b) antibodies were purchased from Cell Signaling (Cambridge, UK).

Techniques: Construct, Transduction, Sequencing, Binding Assay, Purification, FLAG-tag, Negative Control, Control, Inhibition, Activity Assay

Journal: Pharmaceuticals

Article Title: The Inhibition of Stat5 by a Peptide Aptamer Ligand Specific for the DNA Binding Domain Prevents Target Gene Transactivation and the Growth of Breast and Prostate Tumor Cells

doi: 10.3390/ph6080960

Figure Lengend Snippet: Inhibition of Stat5 DNA-binding by S5-DBD-PA. ( a ) Extracts of modified HeLa cells (B9-HeLa) were used for measuring the Stat5 DNA-binding activity by gelshift and luciferase reporter assays. Tyrosine phosphorylated Stat5-dimers are rapidly induced in these cells by Prl stimulation due to a stable overexpression of Stat5 and the PrlR. ( b ) Evaluation of Stat5 DNA binding activity by electrophoretic mobility shift assay (EMSA). For assessing the therapeutic potential of S5-DBD-PA after being taken up by target cells, Stat5 inducible B9-HeLa cells were treated with recombinant S5-DBD-PA and the non-specific hTRX-scaffold control protein for 4 h in the indicated concentrations (experiment on the left). Stat5 was activated subsequently by adding 100 ng/mL Prl. Untransduced membrane bound proteins were removed by acid-wash prior to lysate preparation. Three µg of the cell lysates were mixed with P 32 -labelled double strand oligonucleotides representing a Stat5 response element of the β-casein promoter. The reaction was carried out at room temperature for 30 min in a binding buffer. Lysates of uninduced or/and untreated cells were used as control. To underline the Stat5 mediated DNA shift a control oligonucleotide and a Stat5 antibody (supershift) was added. Another experiment was done without preincubation of the cells with the recombinant constructs (experiment on the right). B9-HeLa cells were stimulated for 30 min with Prl directly and lysates were prepared. 10 µg of the lysates were incubated for 2 h with 5 µg of recombinant S5-DBD-PA or hTRX under shaking at room temperature. 3 µg of the protein mixtures and 3 µg of untreated B9-HeLa cell lysates (−/+ Prl) were used for the assay. ( c ) Measurement of Stat5-regulated luciferase expression with the dual luciferase reporter assay. B9-HeLa cells were co-transfected with a firefly luciferase Stat5-reporter and a renilla luciferase control reporter to improve the accuracy of the measurement. After 2 days cells were treated for 4 h either with S5-DBD-PA or the scaffold control construct (hTRX) in the indicated concentrations or with the same volume of solvent (dialysis buffer) or PBS. Stat5 was induced by adding 100 ng/mL Prl and luciferase activity was detected after further 4 h. (n = 3; Ø ± SD) Significantly reduced luciferase activities in comparison to PBS treated cells are indicated. ** p < 0.01 (1-way-ANOVA with Bonferroni correction) ( d ) Stat5 DNA-binding is inhibited by intracellular S5-DBD-PA expression. STAT5 inducible B9-HeLa cells were transduced with the pSiEW lentiviral vector expressing either S5-DBD-PA, hTRX or the empty vector. 10 days after infection STAT5 was activated by adding 100 ng/mL Prl and cellular lysates were taken. 4µg of the cell lysates were analyzed by EMSA as described before. As controls a Stat5 supershift and lysates of untreated B9-HeLa cells (−/+ Prl) were used.

Article Snippet: P-STAT5 (C11C5, *9359: Tyr694 Stat5a; Tyr699 Stat5b) and P-STAT5-Alexa Fluor ® 647 (C71E5, *9365: Tyr694 Stat5a; Tyr699 Stat5b) antibodies were purchased from Cell Signaling (Cambridge, UK).

Techniques: Inhibition, Binding Assay, Modification, Activity Assay, Luciferase, Over Expression, Electrophoretic Mobility Shift Assay, Recombinant, Control, Membrane, Construct, Incubation, Expressing, Reporter Assay, Transfection, Solvent, Comparison, Transduction, Plasmid Preparation, Infection

Journal: Pharmaceuticals

Article Title: The Inhibition of Stat5 by a Peptide Aptamer Ligand Specific for the DNA Binding Domain Prevents Target Gene Transactivation and the Growth of Breast and Prostate Tumor Cells

doi: 10.3390/ph6080960

Figure Lengend Snippet: S5-DBD-PA interferes with the nuclear translocation of Stat5 upon prolactin induction. ( a ) Stat5-activation in T-47D breast cancer cells stimulated with Prl. ( b ) Immunofluorescent images of lentiviral transduced T-47D cells either expressing S5-DBD-PA or hTRX were taken 7 days after infection by confocal laser scanning microscopy in the absence or presence of Prl. Cells were stained with a Flag-tag antibody, marked with a Alexa 546 conjugated secondary antibody, and a Alexa 647 conjugated antibody recognizing tyrosine phosphorylated Stat5a and Stat5b. Nuclear staining was performed with DAPI and fluorescence marker (eGFP) expression of the SiEW-lentiviral transfer vector was monitored.

Article Snippet: P-STAT5 (C11C5, *9359: Tyr694 Stat5a; Tyr699 Stat5b) and P-STAT5-Alexa Fluor ® 647 (C71E5, *9365: Tyr694 Stat5a; Tyr699 Stat5b) antibodies were purchased from Cell Signaling (Cambridge, UK).

Techniques: Translocation Assay, Activation Assay, Expressing, Infection, Confocal Laser Scanning Microscopy, Staining, FLAG-tag, Fluorescence, Marker, Plasmid Preparation

Journal: Pharmaceuticals

Article Title: The Inhibition of Stat5 by a Peptide Aptamer Ligand Specific for the DNA Binding Domain Prevents Target Gene Transactivation and the Growth of Breast and Prostate Tumor Cells

doi: 10.3390/ph6080960

Figure Lengend Snippet: S5-DBD-PA suppresses the viability and the growth of tumor cells. ( a ) A431 epidermal, PC-3 prostate and HCT116 colorectal carcinoma cell lines were infected with SiEW-lentiviral vectors encoding either S5-DBD-PA, hTRX or the empty vector. Over a period of 20 days cell viability and concomitant growth was monitored by XTT assay. Results are shown as the percentage of viable cells compared to the mock-treated control. Stat5 and transgene expression was verified by western blot 7 days after infection using antibodies recognizing either total or tyrosine phosphorylated Stat5 protein and a flag tag antibody. ( b ) The same experiments were done with unstimulatedT-47D breast cancer cells. Protein expression was analyzed after 7 days with and without 30 min Prl stimulation. (n = 3; Ø ± SD). Significantly reduced XTT-values in comparison to empty vector expressing cells are indicated. * p < 0.05, ** p < 0.01, *** p < 0.001 (2-way-ANOVA with Bonferroni correction).

Article Snippet: P-STAT5 (C11C5, *9359: Tyr694 Stat5a; Tyr699 Stat5b) and P-STAT5-Alexa Fluor ® 647 (C71E5, *9365: Tyr694 Stat5a; Tyr699 Stat5b) antibodies were purchased from Cell Signaling (Cambridge, UK).

Techniques: Infection, Plasmid Preparation, XTT Assay, Control, Expressing, Western Blot, FLAG-tag, Comparison