Journal: Nucleic Acids Research

Article Title: Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling

doi: 10.1093/nar/gkr978

Figure Lengend Snippet: Identification of the minimal human P2P. ( A ) Schematic representation of P2P 5′-truncations and their cloning strategy as used in this study. The translational start site is designated ‘+1’. ( B ) Regulatory DNA regions of the human PHD2 gene were cloned into luciferase reporter vectors that were transiently transfected into human U2OS osteosarcoma cells. One day after transfection, cells were incubated for 24 h at 20 or 0.2% O 2 . Hypoxic IF (mean values ± SD) of relative luciferase activities were calculated from three independent experiments performed in triplicates. Mutation of a single HBS (black rectangles in A) completely abrogated hypoxic inducibility of all constructs. ( C ) HeLa and U2OS cells were incubated at 20 or 0.2% O 2 for 4–24 h and protein levels of HIF-1α, PHD2 and β-actin were analyzed by immunoblotting. Total RNA was isolated from cultures treated as in (B) and mRNA levels of PHD2 and CA9 were determined by RT-qPCR. Transcript levels of CA9 served as positive control to confirm continuous hypoxic responses. Gene expression levels were expressed in relation to ribosomal L28 mRNA (rel. levels) calculated from three independent experiments (±SD).

Article Snippet: Protein concentrations were determined by the Bradford method and 50–80 μg of cellular protein were subjected to immunoblot analysis using the following antibodies: mouse monoclonal antibody (mAb), anti-human HIF-1α (clone 54/HIF-1α; BD Transduction Laboratories), mAb anti-ETV4 [PEA3 ( ); Santa Cruz Biotechnology], rabbit anti-ETV4 (sdix20580002; Novus Biologicals), rabbit anti-human PHD2 (NB100-137; Novus Biologicals), mAb anti-FIH-1 (NBP1-30333; Novus Biologicals), mAb anti-p300 (554215; BD Pharmingen) and mAb anti-β-actin (clone AC-74; Sigma).

Techniques: Cloning, Clone Assay, Luciferase, Transfection, Incubation, Mutagenesis, Construct, Western Blot, Isolation, Quantitative RT-PCR, Positive Control, Gene Expression

Journal: Nucleic Acids Research

Article Title: Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling

doi: 10.1093/nar/gkr978

Figure Lengend Snippet: Hypoxic transactivation of the P2P by ETV4 requires HIF-1α activity. ( A ) Standard dual luciferase reporter gene assays of seven reevaluated hits from the transcription factor overexpression array. Wild-type (left panel) or HBS mutant (right panel) P2P regions controlling firefly luciferase reporter plasmids were cotransfected into U2OS cells together with expression constructs of the aforementioned factors. Transfection of an empty expression vector (empty) served as negative control and differences in transfection efficiency were controlled by cotransfecting SV40 promoter driven renilla luciferase. Cells were cultured at 20% or 0.2% oxygen for 24 h before dual luciferase activities were determined. ( B ) Transient RNAi mediated knock down of HIF-1α fully abrogated hypoxic activation of the P2P by ETV4. U2OS cells were transiently transfected with siRNA oligonucleotides targeting HIF-1α (siHIF1α, right panel) or a control sequence having no human target (siControl, left panel). Reporter gene experiments using the P2P reporter construct with only wild-type HBS were performed as described in (A). The inset shows an immunoblot confirming the robust knock down of HIF-1α in U2OS cells. ( C ) ETV4 and HIF-1 synergism in hypoxic gene activation is not restricted to the P2P. A heterologous hypoxia responsive reporter gene containing two functional HBS from the human Transferrin hypoxia response element (pGL-TfHRE wt) was tested in luciferase reporter assays as described in (A). Mutation of both HBS (pGL-TfHRE mut) caused an abrogation of the signal as seen in (A). ( D and E ) Forced expression of ETV4 in U2OS cells upregulates endogenous PHD2 protein and transcript levels. ( D ) Whole cell lysates were prepared from cells exposed for 16 h to 20 or 0.2% oxygen and analyzed for HIF-1α, ETV4, PHD2 and β-actin levels by immunoblotting. ( E ) Total RNA was extracted of similarly treated cells and mRNA levels of PHD1, PHD2 and L28 were quantified by RT-qPCR. Data are shown in relation to ribosomal L28 mRNA (rel. levels) calculated from three independent experiments (** P < 0.01, paired Student's t -test).

Article Snippet: Protein concentrations were determined by the Bradford method and 50–80 μg of cellular protein were subjected to immunoblot analysis using the following antibodies: mouse monoclonal antibody (mAb), anti-human HIF-1α (clone 54/HIF-1α; BD Transduction Laboratories), mAb anti-ETV4 [PEA3 ( ); Santa Cruz Biotechnology], rabbit anti-ETV4 (sdix20580002; Novus Biologicals), rabbit anti-human PHD2 (NB100-137; Novus Biologicals), mAb anti-FIH-1 (NBP1-30333; Novus Biologicals), mAb anti-p300 (554215; BD Pharmingen) and mAb anti-β-actin (clone AC-74; Sigma).

Techniques: Activity Assay, Luciferase, Over Expression, Mutagenesis, Expressing, Construct, Transfection, Plasmid Preparation, Negative Control, Cell Culture, Knockdown, Activation Assay, Control, Sequencing, Western Blot, Functional Assay, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling

doi: 10.1093/nar/gkr978

Figure Lengend Snippet: Transcriptional cooperation between ETV4 and HIF-1 is disrupted by CITED2. ( A ) Schematic representation of HIF-1α and ETV4 domain structure and fusion constructs used in mammalian two-hybrid assays. PAS, PER-ARNT-SIM; bHLH, basic helix–loop–helix domain; ODD, oxygen-dependent degradation domain; NRR, negative regulatory region; NAD and CAD, amino-carboxy-terminal activation domain and CADs, respectively. A GAL4-DBD was fused to regions encompassing the HIF-1α NAD and CAD. Full-length ETV4 bearing two activation domains (AD, acidic domain; Ct, carboxy-terminal tail) flanking a central ETS domain was fused to a VP16 activation domain (VP16-AD). Numbers indicate the amino acids present in the respective constructs. ( B ) U2OS cells were cotransfected with a Gal4-responsive reporter plasmid and Gal4-HIF-1α (GH1α) constructs alone or in combination with VP16-ETV4. The GH1α fusion constructs are specified by the aminoterminal starting amino acid of the truncated HIF-1α regions (530, 740 and 786, respectively). Following transfection, cells were evenly split and incubated at 20 or 0.2% O 2 before luciferase activities were determined 24 h later. Noninteracting Gal4 DBD-p53 and VP16-AD-CP1 served as negative control (neg. ctrl.), while the interactions between Gal4 DBD-PHD2 and VP16-AD-HIF-2α(ODD) or VP16-AD-FKBP38 were used as positive controls (pos. ctrl. 1 and pos. ctrl. 2, respectively). ( C ) Scheme of the potential interactions between HIF-1, p300/CBP and ETV4 as assessed by mammalian two-hybrid assays. Both CITED2 and FIH can block the interaction between HIF-1α and p300/CBP. ( D ) Cotransfection of the indicated amounts of a CITED2 expression construct together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). ( E ) Cotransfection of siRNA directed against p300 together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). The p300 knock down efficiency of different siP300 oligonucleotides was analyzed by immunoblotting (upper panel) and siP300#1 was chosen for further experiments. ( F ) Cotransfection of siRNA directed against FIH together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). The efficiency of the siFIH mediated FIH knock down was confirmed by immunoblotting as shown in the inset. ( G ) ChIP of normoxic or hypoxic PC3 cells using antibodies directed against HIF-1α or ETV4, or control serum. The amount of coprecipitated chromatin derived from the human P2P region (encoded by EGLN1 ) containing the HBS was determined by PCR followed by agarose gel electrophoresis.

Article Snippet: Protein concentrations were determined by the Bradford method and 50–80 μg of cellular protein were subjected to immunoblot analysis using the following antibodies: mouse monoclonal antibody (mAb), anti-human HIF-1α (clone 54/HIF-1α; BD Transduction Laboratories), mAb anti-ETV4 [PEA3 ( ); Santa Cruz Biotechnology], rabbit anti-ETV4 (sdix20580002; Novus Biologicals), rabbit anti-human PHD2 (NB100-137; Novus Biologicals), mAb anti-FIH-1 (NBP1-30333; Novus Biologicals), mAb anti-p300 (554215; BD Pharmingen) and mAb anti-β-actin (clone AC-74; Sigma).

Techniques: Construct, Activation Assay, Plasmid Preparation, Transfection, Incubation, Luciferase, Negative Control, Blocking Assay, Cotransfection, Expressing, Activity Assay, Knockdown, Western Blot, Control, Derivative Assay, Agarose Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling

doi: 10.1093/nar/gkr978

Figure Lengend Snippet: Genome-wide microarray expression analysis reveals a broad role for ETV4 in HIF mediated hypoxic gene regulation. ( A ) Efficient knock down of ETV4 in human PC3 prostate cancer cells. PC3 cells were stably transduced with lentiviral shRNA expression vectors encoding either a nontarget control (shNTC) or shETV4. Following 24 h of exposure to 20% O 2 or 0.2% O 2 , ETV4, HIF-1α, PHD2 and β-actin protein levels were analyzed by immunoblotting. ( B ) Total RNA was isolated from cultures treated as in (A) and mRNA levels of ETV4 and its target gene COX2 were determined by RT-qPCR. Gene expression levels were expressed in relation to ribosomal L28 mRNA (rel. levels) calculated from three independent experiments. ( C ) Venn diagram showing the number of transcripts regulated by either an at least twofold induction by hypoxia alone (red), an at least twofold reduction in normoxic cells by the knock down of ETV4 (green), or an at least twofold reduction in hypoxic cells by the knock down of ETV4 (blue), respectively. ( D ) Heatmap of the individual expression levels of the 47 transcripts that required ETV4 for efficient hypoxic induction. ( E ) Expression levels of four randomly chosen transcripts shown in (D) were confirmed by RT-qPCR as described for (B).

Article Snippet: Protein concentrations were determined by the Bradford method and 50–80 μg of cellular protein were subjected to immunoblot analysis using the following antibodies: mouse monoclonal antibody (mAb), anti-human HIF-1α (clone 54/HIF-1α; BD Transduction Laboratories), mAb anti-ETV4 [PEA3 ( ); Santa Cruz Biotechnology], rabbit anti-ETV4 (sdix20580002; Novus Biologicals), rabbit anti-human PHD2 (NB100-137; Novus Biologicals), mAb anti-FIH-1 (NBP1-30333; Novus Biologicals), mAb anti-p300 (554215; BD Pharmingen) and mAb anti-β-actin (clone AC-74; Sigma).

Techniques: Genome Wide, Microarray, Expressing, Knockdown, Stable Transfection, Transduction, shRNA, Control, Western Blot, Isolation, Quantitative RT-PCR, Gene Expression

Journal: Nucleic Acids Research

Article Title: Synthetic transactivation screening reveals ETV4 as broad coactivator of hypoxia-inducible factor signaling

doi: 10.1093/nar/gkr978

Figure Lengend Snippet: Role of ETV4 in the regulation of established HIF target genes in vitro and in vivo . ( A ) Dot plots showing the correlation between transcripts in normoxic versus hypoxic control cells (left panel) or in hypoxic control versus hypoxic ETV4 knock down cells (right panel) as derived from the gene array data (grey dots). Red dots refer to internal controls and the blue dot shows ETV4 which is downregulated in shETV4 cells. Green dots indicate the positions of a predefined set of 61 well-established HIF target genes. ( B ) Heat map of the 61 HIF target genes ranked by the magnitude of ETV4 requirement for hypoxic induction according to differences in hypoxic expression levels with Δhyp = log 2 (shNTC_hypoxia) − log 2 (shETV4_hypoxia) and mean hypoxic expression levels centered to the mean of normoxic control cells. ( C ) Exemplary mRNA levels of HIF target genes which either require ETV4 for efficient hypoxic induction (PHD3 and CA9) or which remain unaffected by the ETV4 knock down (GLUT1 and PAI1). mRNA was quantified as described for B. ( D and E ) Correlation between ETV4 and established markers for tissue hypoxia in human breast cancer. ( D ) Independent specimens (spec.) of immunohistochemical evaluation of ETV4 expression in primary mammary carcinoma with high (upper panel) or low (lower panel) ETV4 expression levels. ( E ) Rank-order correlations (Spearman's rho) for ETV4 and PHD2 as well as known markers reflecting tissue hypoxia (HIF-1α, HIF-2α, PAI1, GLUT1 and CA9) are summarized in a cross table. The number of cases where both of the correlated markers could be assessed is displayed in parentheses. Asterisks indicate statistical significance with * P < 0.05 and ** P < 0.01.

Article Snippet: Protein concentrations were determined by the Bradford method and 50–80 μg of cellular protein were subjected to immunoblot analysis using the following antibodies: mouse monoclonal antibody (mAb), anti-human HIF-1α (clone 54/HIF-1α; BD Transduction Laboratories), mAb anti-ETV4 [PEA3 ( ); Santa Cruz Biotechnology], rabbit anti-ETV4 (sdix20580002; Novus Biologicals), rabbit anti-human PHD2 (NB100-137; Novus Biologicals), mAb anti-FIH-1 (NBP1-30333; Novus Biologicals), mAb anti-p300 (554215; BD Pharmingen) and mAb anti-β-actin (clone AC-74; Sigma).

Techniques: In Vitro, In Vivo, Control, Knockdown, Derivative Assay, Expressing, Immunohistochemical staining

Journal: Cell

Article Title: Kinetochore microtubule dynamics and attachment stability are regulated by Hec1.

doi: 10.1016/j.cell.2006.09.047

Figure Lengend Snippet: Figure 1. Epitope Mapping and Cellular Localization of Hec1 Monoclonal Antibody 9G3 (A) Peptides covering the sequence of human Hec1 were adsorbed onto nitrocellulose and immunoprobed with 9G3. As a control, HeLa extract was adsorbed onto the nitrocellulose at region H-12. Both the control spot and spot C-2 (amino acids 200–215) were positively identified. (B) Representation of the Ndc80 complex as predicted from previous publications (Wei et al., 2005; Ciferri et al., 2005). The asterisk marks the site on Hec1 where 9G3 binds. (C and D) Localization of 9G3 (green) and an antibody to Spc24 (red) in PtK1 cells (C) and HeLa cells (D). Linescans were carried out on sister kinet- ochore pairs from both HeLa cells (n = 40 pairs/3 cells) and PtK1 cells (n = 34 pairs/4 cells), and in all cases Hec1 localized exteriorly to Spc24 at kinetochores. (E) Western blot of whole-cell PtK1 extract with 9G3 as a probe. (F) Immunofluorescent image of a PtK1 cell injected with 9G3. To the right of each cell panel in (C), (D), and (F), a single kinetochore pair has been enlarged. The graphs represent the linescan data from the single kinetochore pair. Scale bars in (C), (D), and (F) = 5 mm.

Article Snippet: Hec1 Antibody Epitope Mapping A peptide array (containing peptides of 15 amino acids in length with a 7 amino acid overlap) covering the entire human Hec1 sequence was generated (New England Peptide, Gardner, MA).

Techniques: Sequencing, Control, Western Blot, Injection

Journal: Cell

Article Title: Kinetochore microtubule dynamics and attachment stability are regulated by Hec1.

doi: 10.1016/j.cell.2006.09.047

Figure Lengend Snippet: Figure 4. Loss of Kinetochore Oscillations and Plus-End MT Polymerization in Hec1 9G3-Injected Cells (A and B) Kinetochore behavior was analyzed by live-cell fluorescence timelapse imaging. Cells were injected with rhodamine-labeled tubulin and Alexa 488-conjugated CENP-F antibodies (A), or additionally with 9G3 (B). Images were acquired every 15 s. Selected planes are shown from the timelapse sequences (A and B, top). Selected kinetochore pairs are boxed and a time series of 12 images for each pair is shown below (A and B, bottom). Kinetochores from the control cell exhibited oscillatory behavior and periods of stretching and relaxation (example in A, bottom), whereas kinetochores from the 9G3-injected cell did not oscillate (example in B, bottom). (C and D) EB1-GFP-expressing PtK1 cells were injected with Texas Red dextran alone (C) or in combination with 9G3 (D). Images were acquired every 10 s. A region containing a kinetochore pair and the spindle poles was extracted from the timelapse sequence and shown to the right. The bright spots in extracted images are spindle poles. (E) A buffer-injected monopolar cell exhibits chromosome oscillations toward and away from the pole both prior to and after injection (top panel). In cells injected with 9G3, chromosomes stopped oscillating and moved poleward after injection (middle and bottom panels). In all panels, scale bars = 5 mm.

Article Snippet: Hec1 Antibody Epitope Mapping A peptide array (containing peptides of 15 amino acids in length with a 7 amino acid overlap) covering the entire human Hec1 sequence was generated (New England Peptide, Gardner, MA).

Techniques: Injection, Imaging, Labeling, Control, Expressing, Sequencing

Journal: Cell

Article Title: Kinetochore microtubule dynamics and attachment stability are regulated by Hec1.

doi: 10.1016/j.cell.2006.09.047

Figure Lengend Snippet: Figure 6. Aurora B Kinase Phosphoryla- tion and Regulation of Hec1 (A) Left: Aurora B/INCENP790–856 in vitro kinase assay with Histone H3 as a control substrate (lanes 1 and 2) and Hec11–230 (lanes 3 and 4). Antibody 9G3 was added to the reaction mixtures in lanes 2 and 4. Right: Normalized quantification of radioactive phosphate for lanes 1–4. Molecular weight standards are indi- cated in kilodaltons. (B) PtK1 cells were transfected with WT-GFP- Hec1 (upper row) or mutant 6A-GFP-Hec1 (bot- tom three rows) for 40 hr prior to fixation for im- munofluoresence. Scale bar = 5 mm. (C) Cells transfected with WT-GFP-Hec1 and 6A-GFP-Hec1 were scored for chromosome alignment and assigned to one of three cate- goryies: chromosomes all aligned, chromo- somes mostly aligned (1–2 chromosomes off the metaphase plate), or chromosomes mostly unaligned (fewer than 3 aligned chromosomes) (n = 52 WT-GFP-Hec1 cells; n = 40 6A-GFP- Hec1 cells). (D) Cells were scored for merotelic kineto- chores. For WT-GFP-Hec1-expressing cells: n = 19 prometaphase cells, n = 31 metaphase/ near metaphase cells, and n = 11 anaphase cells. For 6A-GFP-Hec1-expressing cells: n = 47 prometaphase cells, n = 13 metaphase/ near metaphase cells, and n = 18 anaphase cells.

Article Snippet: Hec1 Antibody Epitope Mapping A peptide array (containing peptides of 15 amino acids in length with a 7 amino acid overlap) covering the entire human Hec1 sequence was generated (New England Peptide, Gardner, MA).

Techniques: In Vitro, Kinase Assay, Control, Molecular Weight, Transfection, Mutagenesis, Expressing

Journal: Cell

Article Title: Kinetochore microtubule dynamics and attachment stability are regulated by Hec1.

doi: 10.1016/j.cell.2006.09.047

Figure Lengend Snippet: Figure 7. Model for Hec1 Regulation of kMT Dynamics and Attachment (A) Mitotic spindle arrangement in a control cell (top) in which normal Aurora B phophorylation and dephosphorylation occur. kMT plus ends exhibit dynamic instability and undergo periods of attachment and detachment. Net polymerization at plus ends of kMTs is balanced by net depolymerization at minus ends. After addition of 9G3 (bottom), the N terminus of Hec1 can no longer be phosphorylated by Aurora B, and kMT detachment and dy- namic instability are suppressed. Minus-end depolymerization is not inhibited, and active depolymerases shorten kinetochore fibers. Hyper-stretch of centromeres arises from pulling forces exerted by the centrosome-associated minus-end organizing complexes as they maintain connection with the depolymerizing minus ends of the kinetochore fibers. (B) Regulation of kMT plus-end dynamic instability and attachment strength at three possible interfaces. Interface 1 is between the N terminus of Hec1 and the MT lattice; interface 2 is between the N terminus of Hec1 and a kinetochore-binding MAP, and interface 3 is between the MAP and the MT lattice (see text for details).

Article Snippet: Hec1 Antibody Epitope Mapping A peptide array (containing peptides of 15 amino acids in length with a 7 amino acid overlap) covering the entire human Hec1 sequence was generated (New England Peptide, Gardner, MA).

Techniques: Control, De-Phosphorylation Assay, Binding Assay

Journal: Brain, behavior, and immunity

Article Title: Identification of the Antigenic Epitopes of Maternal Autoantibodies in Autism Spectrum Disorders

doi: 10.1016/j.bbi.2017.12.014

Figure Lengend Snippet: Characterization of maternal plasma samples used within the discovery microarrays.

Article Snippet: These cut-off values were selected based on cutoff ranges reported in similar autoantibody epitope mapping studies that also used PEPperPRINT microarray technology ( Hamilton et al., 2015 ; Korkmaz et al., 2013 ).

Techniques: Clinical Proteomics, Microarray, Diagnostic Assay

Journal: Brain, behavior, and immunity

Article Title: Identification of the Antigenic Epitopes of Maternal Autoantibodies in Autism Spectrum Disorders

doi: 10.1016/j.bbi.2017.12.014

Figure Lengend Snippet: Representative discovery array with LDH-A, STIP1 and CRMP1 15-mer amino acid sequences probed with a maternal sample positive for all three proteins. Each box shows the region of antibody reactivity (Box 1 = LDH-A; Box 2 = STIP1; Boxes 3 and 4 = CRMP1). The microarray frames (controls) in each were stained with anti-Flag (shown in green) and anti-HA antibodies (shown in red), while the red spots within the array represent areas of antibody recognition.

Article Snippet: These cut-off values were selected based on cutoff ranges reported in similar autoantibody epitope mapping studies that also used PEPperPRINT microarray technology ( Hamilton et al., 2015 ; Korkmaz et al., 2013 ).

Techniques: Microarray, Staining

Journal: Brain, behavior, and immunity

Article Title: Identification of the Antigenic Epitopes of Maternal Autoantibodies in Autism Spectrum Disorders

doi: 10.1016/j.bbi.2017.12.014

Figure Lengend Snippet: Screening microarray - proportion of maternal reactivity to individual peptides

Article Snippet: These cut-off values were selected based on cutoff ranges reported in similar autoantibody epitope mapping studies that also used PEPperPRINT microarray technology ( Hamilton et al., 2015 ; Korkmaz et al., 2013 ).

Techniques: Microarray