Journal: Endocrinology

Article Title: PROX1 Promotes Secretory Granule Formation in Medullary Thyroid Cancer Cells.

doi: 10.1210/en.2015-1973

Figure Lengend Snippet: Figure 5. Effect of PROX1 on known CHGA transcription-regulatory factors. A, A schematic diagram of the CHGA promoter region and the putative PROX1 binding site. CRE, cAMP-responsive element: ex, exon; RE1, repressor element 1; TSS, transcription start site. ChIP-PCR target indicates the region amplified in the chromatin immunoprecipitation- based PCR assay. B, Semiquantitative RT-PCR analysis of PROX1 and CHGA, REST mRNA expression, and Western blot analysis of REST, CREB, phosphorylated CREB (CREB-P), and -actin expression in PROX1-inducible KTC1 and TTA1 cells in the absence (0 h) or presense (12, 24, and 48 h) of the inducer Dox. Data are shown as means SD. C, ChIP-PCR assay of CREB and REST binding to the CHGA gene. Cell lysates of Dox-treated or untreated FLAG-tagged PROX1-transfected KTC1 cells (KTC1-PROX1) were immunoprecipitated by using anti- CREB or anti-PROX1 antibody or normal rabbit IgG. Coprecipitation of gene fragments of CHGA containing CRE or RE1 sequence (CHGA[481 to 367] or CHGA[272 to 808], respectively) were assayed using PCR. CREB, rabbit anti-CREB antibody; IgG, normal rabbit IgG; IP, immunoprecipitation; REST, rabbit anti-REST antibody. The experiment was conducted in triplicate. Data are shown as means SD.

Article Snippet: Briefly, the chromatin solution was incubated with 1 g or recommended amount of mouse anti-FLAG antibody (Sigma-Aldrich), rabbit anti-CREB (Cell Signaling Technology), rabbit anti-REST antibody (Bethyl Laboratories, Inc), normal mouse IgG (Santa Cruz Biotechnology), or normal rabbit IgG (Santa Cruz Biotechnology), and the antibody/chromatin mixtures were then precipitated with Protein G beads.

Techniques: Binding Assay, Amplification, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Transfection, Immunoprecipitation, Sequencing

Journal: Cell Death Discovery

Article Title: Cigarette smoke activates the parthanatos pathway of cell death in human bronchial epithelial cells

doi: 10.1038/s41420-019-0205-3

Figure Lengend Snippet: PARP–1 recognition of smoke-mediated DNA strand breaks activates itself to poly-adenosylate (PAR) itself and other nuclear proteins . Extensive DNA damage resulting in PARP–1 overactivation produces branched chains of PAR . Poly (ADP-ribose) glycohydrolase (PARG) releases PAR by cleavage that unless hydrolyzed by ADP-ribosylhydrolase 3 (ARH3) induces mitochondrial proteins Apoptosis-Inducing Factor (AIF) and EndonucleaseG (EndoG) to translocate to the nucleus causing genomic DNA fragmentation

Article Snippet: Samples were amplified on a CFX-96 Bio-Rad real time PCR machine using SsoAdvanced universal probes supermix (172–5284, Bio-Rad) and TaqMan primers for PARP-1 and GAPDH as housekeeping gene (Hs00242302_m1 resp. Hs02758991_g1, Thermo Fisher Scientific).

Techniques:

Journal: Cell Death Discovery

Article Title: Cigarette smoke activates the parthanatos pathway of cell death in human bronchial epithelial cells

doi: 10.1038/s41420-019-0205-3

Figure Lengend Snippet: a RT-PCR of PARP-1 normalized to GAPDH in HBE cells from non-smokers ( n = 12) and smokers ( n = 17). b Western blot analysis of PARP-1 protein expression normalized to β-actin in HBE cells from non-smokers ( n = 6) and smokers ( n = 12). Plotted individual data points with line representing mean

Article Snippet: Samples were amplified on a CFX-96 Bio-Rad real time PCR machine using SsoAdvanced universal probes supermix (172–5284, Bio-Rad) and TaqMan primers for PARP-1 and GAPDH as housekeeping gene (Hs00242302_m1 resp. Hs02758991_g1, Thermo Fisher Scientific).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: REST Regulates DYRK1A Transcription in a Negative Feedback Loop *

doi: 10.1074/jbc.M110.174540

Figure Lengend Snippet: REST activates human DYRK1A gene transcription. A, pDYluc-A contains the functional promoter of DYRK1A gene. The promoter construct pDYluc-A, containing the 1127-bp fragment from the human DYRK1A gene 5′-UTR, was transfected into HEK293 cells. Luciferase activity was measured 24 h after transfection by a luminometer. The values represent the means ± S.E. (n = 3). *, p < 0.001 by Student's t test. B, DYRK1A gene promoter is activated by REST. REST was co-transfected with pDYluc-A into HEK293 cells. Luciferase assay was performed 24 h after transfection. The values represent the means ± S.E. (n = 3). *, p < 0.001 by Student's t test. C, RT-PCR showed that REST knockdown construct psiREST can knock down Rest mRNA expression. β-Actin was used as the internal control. The values represent the means ± S.E. (n = 3). *, p = 0.0006 by Student's t test. D, the knockdown effect of psiREST was verified in protein level by Western blot. HEK293 cells were co-transfected with REST expression plasmid and psiREST. REST was detected with anti-REST antibody. β-Actin was used as loading control. The values represent the means ± S.E. (n = 3). *, p = 0.04 by Student's t test. E, RT-PCR showed that BDNF mRNA was repressed by REST overexpression in HEK293 cells. β-Actin was amplified as the internal control. *, p < 0.001 by Student's t test. F, RT-PCR showed that REST overexpression significantly increased DYRK1A mRNA, whereas REST knockdown significantly decreased DYRK1A mRNA levels. β-Actin was amplified as the internal control. The values represent the means ± S.E. (n = 3). *, p < 0.001 by Student's t test. G, real time PCR showed that DYRK1A expression was elevated with REST overexpression. TaqMan gene expression assay of DYRK1A was performed with an ABI 7900HT real time PCR system. 18 S rRNA was chosen for the internal control. The values represent the means ± S.E. (n = 4). *, p = 0.0032 by Student's t test. Con, control.

Article Snippet: The slices were immunostained with rabbit anti-DYRK1A (1:25; Cell Signaling Technology, Danvers, MA), rabbit anti-REST (1:500; Upstate), or mouse anti-MAP-2 (1:100; Vector Laboratories, Burlingame, CA) primary antibodies at 4 °C overnight.

Techniques: Functional Assay, Construct, Transfection, Luciferase, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Plasmid Preparation, Over Expression, Amplification, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: REST Regulates DYRK1A Transcription in a Negative Feedback Loop *

doi: 10.1074/jbc.M110.174540

Figure Lengend Snippet: Identification of a functional NRSE site in human DYRK1A promoter. A, schematic diagram of the human DYRK1A promoter deletion constructs consisting of a 5′-flanking region with serial deletions cloned into pGL3-Basic in front of the luciferase gene (Luc). Arrowheads indicate the directions of transcription. The numbers represent the end points of each construct. The positions of three putative NRSE sites are shown at the bottom. B, the deletion plasmids were confirmed by sequencing and restriction enzyme digestion checking on a 1.5% agarose gel. Vector size is 4.7 kb, and the DYRK1A gene 5′-flanking fragment inserts range from 0.5 to 1.5 kb. C, the deletion plasmids were transfected into REST-competent C6 or REST-incompetent PC12 cells. Plasmid pRL-TK was used to normalize the transfection efficiency, and luciferase activity was measured at 24 h by a luminometer. The values represent the means ± S.E. (n = 5). *, p < 0.001 by Student's t test. D, PC12 cells were co-transfected with REST expression vector and various DYRK1A promoter deletion constructs. Luciferase assay was performed 24 h after transfection. The values represent the means ± S.E. (n = 5). *, p < 0.001 by Student's t test. E, pDYlucm was made to contain the mutant NRES site of DYRK1A promoter. The mutant plasmid, as well as the wild type pDYluc-A, was co-transfected with REST expression plasmid into HEK293 cells. Plasmid pRL-TK was used to normalize the transfection efficiency, and luciferase activity was measured at 24 h by a luminometer. The values represent the means ± S.E. (n = 3). *, p < 0.01 by Student's t test. F and G, EMSA was performed with IRDye 700-labeled consensus NRSE oligonucleotides (F) or NRSE site of DYRK1A promoter (G). First lane, labeled probe without nuclear extract. Second lane, incubation of infrared labeled NRSE probe with HeLa nuclear extracts retarded the migration rate of the labeled probe, which formed a new shifted DNA-protein complex band. The addition of anti-REST antibody further shifted the band to a larger mass shown by a longer running time (third lane in the lower panel of G). Competition assays were performed by further adding different concentrations of molar excess of unlabeled competitive oligonucleotides. H, anti-REST antibody was used to immunoprecipitate the cross-linked REST-DNA complex in ChIP assay in SH-SY5Y cells. A pair of primers targeting DYRK1A was used to amplify NRSE-DY1 and NRSE-DY3. Signals amplified from input were used as size markers. Mouse IgG was used as a negative control. Con, control.

Article Snippet: The slices were immunostained with rabbit anti-DYRK1A (1:25; Cell Signaling Technology, Danvers, MA), rabbit anti-REST (1:500; Upstate), or mouse anti-MAP-2 (1:100; Vector Laboratories, Burlingame, CA) primary antibodies at 4 °C overnight.

Techniques: Functional Assay, Construct, Clone Assay, Luciferase, Sequencing, Agarose Gel Electrophoresis, Plasmid Preparation, Transfection, Activity Assay, Expressing, Mutagenesis, Labeling, Incubation, Migration, Amplification, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: REST Regulates DYRK1A Transcription in a Negative Feedback Loop *

doi: 10.1074/jbc.M110.174540

Figure Lengend Snippet: DYRK1A and REST coordinately expressed in mouse brain during neurodevelopment and in cell lines. A and B, immunofluorescence staining was performed on coronal slices from adult mouse brain. A Cy3-conjugated anti-rabbit secondary antibody was used to visualize DYRK1A (A1 and B1, red) and REST (A4 and B5, red). MAP2 (microtubule-associated protein 2) was stained to serve as a neuronal marker. A fluorescein-conjugated anti-mouse secondary antibody was used to visualize MAP2 (B2 and B6, green). The nuclei were counterstained with blue DAPI (A2, A5, B3, and B7). Overlay showed that both DYRK1A and REST are enriched in neurons in hippocampus (A3 and A6) and cerebral cortex (B4 and B8). The images were captured with a Leica fluorescence microscope at ×400 (A) or ×600 (B). C, quantitative RT-PCR was performed on cDNA templates prepared from normal mouse brain aging at embryonic days 13 (E13) and 17 (E17); at P1, P7, P14, and P30; and in adult mice. One to three mice were used in each time point as indicated by the numbers after the hyphens. p = 0.0028 by correlation test. Rest (D) and Dyrk1a (E) mRNA levels were determined by RT-PCR in C6, N2A, and PC12 cells. β-Actin was amplified as the internal control. F, quantification of D and E showed that Rest and Dyrk1a were coordinately expressed at low levels in PC12 cells and at high levels in N2a and C6 cells. The values represent the means ± S.E. (n = 3). *, p = 0.0002 by correlation test.

Article Snippet: The slices were immunostained with rabbit anti-DYRK1A (1:25; Cell Signaling Technology, Danvers, MA), rabbit anti-REST (1:500; Upstate), or mouse anti-MAP-2 (1:100; Vector Laboratories, Burlingame, CA) primary antibodies at 4 °C overnight.

Techniques: Immunofluorescence, Staining, Marker, Fluorescence, Microscopy, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: The Journal of Biological Chemistry

Article Title: REST Regulates DYRK1A Transcription in a Negative Feedback Loop *

doi: 10.1074/jbc.M110.174540

Figure Lengend Snippet: REST expression is down-regulated by DYRK1A protein imbalance. A, psi-DY (siRNA against DYRK1A) was co-transfected with DYRK1A expression plasmid into N2a cells. DYRK1A was detected by anti-FLAG (M2) antibody. β-Actin was used as loading control. The values represent the means ± S.E. (n = 3). *, p < 0.0001 by Student's t test. B, N2a cells were transfected with DYRK1A expression vector pDYRK1A or knockdown vector psi-DY. Rest mRNA expression was detected by RT-PCR. β-Actin was amplified as the internal control. The values represent the means ± S.E. (n = 3). *, p < 0.0001 by Student's t test. C, N2a cells were co-transfected with REST expression vector and pDYRK1A or psi-DY. The cell lysates were separated with 12% SDS-PAGE. REST expression was detected by anti-REST antibody. β-Actin was used as loading control. The values represent the means ± S.E. (n = 3). *, p < 0.01 by Student's t test. D, HEK293 cells co-transfected with REST expression vector, pUbi-His, and pDYRK1A or psi-DY were chased with 50 μg/ml cycloheximide (CHX) for 1 and 2 h. REST expression was detected by anti-REST antibody. β-Actin was used as loading control. E, HEK293 cells co-transfected with REST expression vector and pDYRK1A or psi-DY were lysed and immunoprecipitated with anti-ubiquitin antibody (1:1000) and detected with anti-REST antibody. The values represent the means ± S.E. (n = 3). *, p < 0.01 by Student's t test. F, N2a cells were co-transfected with C terminus REST truncated mutant, FLAG-tagged REST-FS, together with pDYRK1A or psi-DY. REST-FS protein was detected by anti-FLAG (M2) antibody. β-Actin was used as loading control. The values represent the means ± S.E. (n = 3). p > 0.05 by Student's t test. Con, control; IP, immunoprecipitation.

Article Snippet: The slices were immunostained with rabbit anti-DYRK1A (1:25; Cell Signaling Technology, Danvers, MA), rabbit anti-REST (1:500; Upstate), or mouse anti-MAP-2 (1:100; Vector Laboratories, Burlingame, CA) primary antibodies at 4 °C overnight.

Techniques: Expressing, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Amplification, SDS Page, Immunoprecipitation, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: REST Regulates DYRK1A Transcription in a Negative Feedback Loop *

doi: 10.1074/jbc.M110.174540

Figure Lengend Snippet: Transcriptional activity of REST was reduced by DYRK1A protein imbalance. A and B, EMSA showed that REST activity was perturbed by DYRK1A imbalance. N2a cells were transfected with pDYRK1A and psi-DY (A) or co-transfected with REST expression vector (B). EMSA was performed with IRDye 700-labeled consensus NRSE. Nuclear extracts were obtained from N2a cells transfected with pDYRK1A (lane 2), psi-DY (lane 3), or empty vector (lane 4). The specificity of REST-NRSE binding was indicated by competition by consensus NRSE oligonucleotides (lane 5) or NRSE mutant oligonucleotides (lane 6). C and D, N2a cells were transfected with DYRK1A expression vector pDYRK1A or knockdown vector psi-DY. Bdnf mRNA levels were detected by RT-PCR. β-Actin was amplified as the internal control. The values represent the means ± S.E. (n = 3). *, p < 0.001 by Student's t test. E, the BDNF promoter construct pBDNFluc was co-transfected with pDYRK1A or psi-DY. Luciferase assay was performed 24 h after transfection. The values represent the means ± S.E. (n = 3). *, p < 0.001 by Student's t test. F, the DYRK1A promoter construct pDYluc-A was co-transfected with pDYRK1A or psi-DY into N2a cells. Luciferase activity was measured at 24 h by a luminometer. The values represent the means ± S.E. (n = 4). *, p < 0.001 by Student's t test. Con, control.

Article Snippet: The slices were immunostained with rabbit anti-DYRK1A (1:25; Cell Signaling Technology, Danvers, MA), rabbit anti-REST (1:500; Upstate), or mouse anti-MAP-2 (1:100; Vector Laboratories, Burlingame, CA) primary antibodies at 4 °C overnight.

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Labeling, Binding Assay, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Amplification, Construct, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: REST Regulates DYRK1A Transcription in a Negative Feedback Loop *

doi: 10.1074/jbc.M110.174540

Figure Lengend Snippet: Schematic diagram of the cross-interaction of REST and DYRK1A. The imbalance of DYRK1A expression in cells increases the phosphorylation and subsequent ubiquitination of REST protein. Ubiquitinated REST can be degraded by proteasome, which results in the weaker transcription of DYRK1A via a NRSE site located at DYRK1A gene promoter. Thus, a negative feedback loop is formed in DYRK1A gene regulation.

Article Snippet: The slices were immunostained with rabbit anti-DYRK1A (1:25; Cell Signaling Technology, Danvers, MA), rabbit anti-REST (1:500; Upstate), or mouse anti-MAP-2 (1:100; Vector Laboratories, Burlingame, CA) primary antibodies at 4 °C overnight.

Techniques: Expressing

Journal: Nature Communications

Article Title: A neurodegeneration checkpoint mediated by REST protects against the onset of Alzheimer’s disease

doi: 10.1038/s41467-023-42704-6

Figure Lengend Snippet: a Analysis of known (left panels) and de novo motifs (right panels) shows that the REST-RE1 DNA binding motif is highly enriched in the ChIP-seq peaks in both 3xTg and WT mice. b Heat maps showing REST binding to the 507 REST peaks that exhibit significantly higher REST binding in the 3xTg vs. WT cortex. Peak regions are sorted from highest to lowest REST binding levels. c Gene ontology analysis of genes located under the peaks that are predominantly bound in 3xTg mice shows that REST targets genes that regulate cellular metabolism, cell communication, stress responses, and apoptotic cell death. Dashed line: FDR = 0.05. d Examples of REST binding to selected genomic regions in WT (blue: shown is the average of 4 biological replicates) and 3xTg (red: average of 4 biological replicates) mouse cortex resolved by ChIP-seq. The arrows indicate the transcription start sites. The regions within REST ChIP-seq peaks that were amplified by qPCR are indicated in green. e ChIP-qPCR analysis REST binding to peak regions in Cdk5 , Gsk3β , and Daxx genes, in 5- and 11-month-old WT and 3xTg cortex. Also shown is binding by a non-specific IgG control antibody, as well as REST binding to control sites located 10 kb downstream from the REST peaks. n = 3 mice/group. Individual values and the mean ± S.E.M are shown. P -values were generated by one-way ANOVA analyses with Šídák’s multiple comparisons test, followed by Bonferroni correction for the simultaneous testing of 6 genomic regions (see Supplementary Data for all Bonferroni-adjusted P -values). Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used to detect the REST protein: (1) a rabbit polyclonal IgG that recognizes a region between residues 1050–1097 (C-terminus) of REST (Bethyl laboratories, IHC-00141). (2) The REST C-terminal antibody used for ChlP-seq was a gift from Gail Mandel (Vollum Institute).

Techniques: Binding Assay, ChIP-sequencing, Amplification, Generated

Journal: Nature Communications

Article Title: A neurodegeneration checkpoint mediated by REST protects against the onset of Alzheimer’s disease

doi: 10.1038/s41467-023-42704-6

Figure Lengend Snippet: a Cultured human SHY-5Y neuroblastoma cells were transduced with recombinant lentiviruses leading to either REST inhibition (short hairpin RNA; sh4) or overexpression (human REST cDNA: hREST ), as previously described . Expression of γ-secretase components was assessed by qRT-PCR. b REST lx/lx MEF cells were transduced with Cre recombinase (generating REST-KO lines 1–3) or a control vector without Cre (lines WT 1–3). The REST floxed ( REST lx ) and Cre-recombined REST ( REST rec ) alleles were detected by PCR. No REST lx band was detected in REST-KO cells, suggesting complete Cre -mediated recombination. c qRT-PCR using two sets of primers spanning the transcript shows loss of REST mRNA in REST-KO MEFs. d Immunolabeling with anti-REST antibody (REST14 ; white) shows loss of REST expression in REST-KO MEFs. Nuclei are labeled with DAPI (blue). Scale bar, 40 μm. e Western blot analysis of γ-secretase components in WT and REST-KO MEFs. The transferrin receptor (TfR) served as loading control. f Quantification of protein levels normalized to TfR shown as percentage expression in REST-KO relative to WT cells (interrupted line: 100%). For PS1, similar results were seen with antibodies against the N-terminus (NTF; antibody 231f) or C-terminus (CTF; antibody EP2000Y). g REST-KO MEFs show elevated γ-secretase enzymatic activity. Solubilized membranes were incubated with Met-C99-FLAG in the presence or absence of a γ-secretase inhibitor (+I), and levels of AICD-FLAG were determined by Western blotting using TfR as a loading control. h Quantification of γ-secretase activity in membrane preparations from WT and REST-KO cells. AICD-FLAG levels were normalized to TfR, and shown as percentage expression in REST-KO relative to WT cells (interrupted line: 100%). Loss of REST in REST-KO MEFs leads to elevated Aβ40 ( i ) and Aβ42 ( j ) levels following transfection of hAPP WT or hAPP Swe . Lentiviral transduction of human REST cDNA (hREST) suppresses Aβ production. Individual values and the mean ± S.E.M are shown for n = 6 ( a ) or n = 3 ( c , f , h , i , j ) independent experiments. P -values are derived from two-tailed unpaired t -tests ( a , c , f , h ) or one-way ANOVA with Tukey’s post-hoc test ( i , j ). Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used to detect the REST protein: (1) a rabbit polyclonal IgG that recognizes a region between residues 1050–1097 (C-terminus) of REST (Bethyl laboratories, IHC-00141). (2) The REST C-terminal antibody used for ChlP-seq was a gift from Gail Mandel (Vollum Institute).

Techniques: Cell Culture, Transduction, Recombinant, Inhibition, shRNA, Over Expression, Expressing, Quantitative RT-PCR, Plasmid Preparation, Immunolabeling, Labeling, Western Blot, Activity Assay, Incubation, Membrane, Transfection, Derivative Assay, Two Tailed Test

Journal: Cancer Research

Article Title: GP369, an FGFR2-IIIb–Specific Antibody, Exhibits Potent Antitumor Activity against Human Cancers Driven by Activated FGFR2 Signaling

doi: 10.1158/0008-5472.can-10-1489

Figure Lengend Snippet: Figure 1. Characterization of human cancer cell lines with FGFR2 amplification. A, FGFR copy number of human cancer cell lines. Quantitative PCR was carried out using primers specific for FGFRs or a reference gene, transketolase (TKT), and copy number was calculated. B, FGFR expression analysis. Quantitative RT-PCR was carried out using primers specific for FGFRs and HPRT. The expression levels were normalized to HPRT. C, FGFR2 cell surface expression by flow cytometry. The cells were incubated with 100 μL of primary FGFR2 antibodies (5 μg/mL) for 1 h, followed by incubation with 1:100 phycoerythrin (PE)-conjugated secondary antibodies (Jackson ImmunoResearch) for 30 min. Analysis of stained cells was performed on a FC500 flow cytometer (Beckman Coulter).

Article Snippet: These findings rt the hypothesis that dysregulated FGFR2 signaling is the many critical tumorigenesis pathways in human s. Targeted therapy using FGFR2 mAb could be benefir patients with aberrantly activated/amplified FGFR2 female (Taco in 1:1 Tumo calipe formu appro of 10 contro the M mice 0.72 m Resea MFMappro 10 ani by i.p. using Resu Chara FGFR 8/7630.pdf by guest on 20 July 2024 ing. rials and Methods nes and reagents O III, HEC-1-A, AN3 CA, SNU-16, and human lung canll lines were acquired from the American Type Culture tion (ATCC).

Techniques: Amplification, Real-time Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Flow Cytometry, Incubation, Staining

Journal: Cancer Research

Article Title: GP369, an FGFR2-IIIb–Specific Antibody, Exhibits Potent Antitumor Activity against Human Cancers Driven by Activated FGFR2 Signaling

doi: 10.1158/0008-5472.can-10-1489

Figure Lengend Snippet: Figure 2. Epitope mapping of GP369. A, specific binding of GP369 to human and mouse FGFR2-IIIb. The extracellular domains of human FGFR2-IIIb, human FGFR2-IIIc, mouse FGFR2-IIIb, and human Jag-1 (control) were expressed as human Fc fusion proteins and captured on anti-hFc Octet biosensors (ForteBio). Sensograms were recorded when the loaded biosensors were incubated with GP369 in Octet sample diluent (ForteBio). B, epitope mapping. Ten overlapping peptides were biotinylated at their NH2 termini and captured on streptavidin Octet biosensors. Sensograms were recorded when peptide- loaded biosensors were incubated with GP369 in Octet sample diluent. C, GP369 binding epitopes. GP369 binding peptides (from amino acid 309 to 345; yellow) mapped to the structure of FGFR2-IIIb (light blue; D2 on top, D3 at the bottom) in complex with FGF10 (red; PDB structure 1NUN; ref. 35). D, peptide sequences used for epitope mapping. The residues in human FGFR2-IIIb that differ from FGFR2-IIIc are in red. The amino acids involved in binding to FGF10 are highlighted in yellow. The peptides that bind to GP369 are denoted with “+” and those that do not bind are denoted with “−”.

Article Snippet: These findings rt the hypothesis that dysregulated FGFR2 signaling is the many critical tumorigenesis pathways in human s. Targeted therapy using FGFR2 mAb could be benefir patients with aberrantly activated/amplified FGFR2 female (Taco in 1:1 Tumo calipe formu appro of 10 contro the M mice 0.72 m Resea MFMappro 10 ani by i.p. using Resu Chara FGFR 8/7630.pdf by guest on 20 July 2024 ing. rials and Methods nes and reagents O III, HEC-1-A, AN3 CA, SNU-16, and human lung canll lines were acquired from the American Type Culture tion (ATCC).

Techniques: Binding Assay, Control, Incubation

Journal: Cancer Research

Article Title: GP369, an FGFR2-IIIb–Specific Antibody, Exhibits Potent Antitumor Activity against Human Cancers Driven by Activated FGFR2 Signaling

doi: 10.1158/0008-5472.can-10-1489

Figure Lengend Snippet: Figure 3. Characterization of FGFR2 antibodies by proliferation assays. A, GP369 suppresses cell proliferation driven by the WT or the COOH-terminally truncated isoform of FGFR2-IIIb or the mutant variants of FGFR2-IIIb. GP369 mAb was added to FDCP-1 cells expressing FGFR2-IIIb, FGFR2-IIIc, a COOH-terminally truncated IIIb isoform, or the S252W and N550K mutant variants in the presence of FGF1 (8 ng/mL) and heparin (5 μg/mL). MTT assays were carried out after 2 d. B, inhibition of the proliferation of SUM52PE cells by GP369. The cells were incubated in reduced serum medium (0.5% FBS) and were either left untreated (medium) or treated with mIgG or GP369 at various concentrations for 5 d. Cell proliferation was assessed by MTT assays. C, inhibition of the FGF7-induced proliferation of MCF7 cells by GP369. The cells were incubated in serum-free medium and were either left untreated (medium) or treated with mIgG or GP369 at 30 μg/mL for 3 d in the absence or presence of FGF7 (25 ng/mL). Cell proliferation was assessed by MTT assays.

Article Snippet: These findings rt the hypothesis that dysregulated FGFR2 signaling is the many critical tumorigenesis pathways in human s. Targeted therapy using FGFR2 mAb could be benefir patients with aberrantly activated/amplified FGFR2 female (Taco in 1:1 Tumo calipe formu appro of 10 contro the M mice 0.72 m Resea MFMappro 10 ani by i.p. using Resu Chara FGFR 8/7630.pdf by guest on 20 July 2024 ing. rials and Methods nes and reagents O III, HEC-1-A, AN3 CA, SNU-16, and human lung canll lines were acquired from the American Type Culture tion (ATCC).

Techniques: Mutagenesis, Expressing, Inhibition, Incubation

Journal: Cancer Research

Article Title: GP369, an FGFR2-IIIb–Specific Antibody, Exhibits Potent Antitumor Activity against Human Cancers Driven by Activated FGFR2 Signaling

doi: 10.1158/0008-5472.can-10-1489

Figure Lengend Snippet: Figure 4. Inhibition of FGFR2 signaling by GP369. A, GP369 suppresses FGF7-induced FGFR2 tyrosine phosphorylation and ERK1/2 phosphorylation in Ba/F3 cells overexpressing FGFR2-IIIb. After 3 h of serum starvation, the cells were treated with PBS (mock) or 5 μg/mL of either mIgG or GP369 for 1 h at 37°C before stimulation by FGF7 (20 ng/mL) and heparin (5 μg/mL) for 15 min. The protein lysates were analyzed by Western blot. B, GP369 blocks FGF7-induced FGFR2 and FRS2 tyrosine phosphorylation and ERK1/2 phosphorylation in SNU-16 cells. The cells were incubated with PBS (mock) or 5 μg/mL of either mIgG or GP369 for 1 h at 37°C before treatment with either heparin (20 μg/mL) or heparin plus FGF7 (30 ng/mL) for 15 min. Western blotting analysis was then carried out. C, downregulation of the FGFR2 protein levels by GP369. SNU-16 cells were treated with 10 μg/mL of either mIgG or GP369. The cells were harvested at various time points. Total levels of FGFR2 and β-tubulin were determined by Western blotting, and densitometric units quantified using Scion Image software.

Article Snippet: These findings rt the hypothesis that dysregulated FGFR2 signaling is the many critical tumorigenesis pathways in human s. Targeted therapy using FGFR2 mAb could be benefir patients with aberrantly activated/amplified FGFR2 female (Taco in 1:1 Tumo calipe formu appro of 10 contro the M mice 0.72 m Resea MFMappro 10 ani by i.p. using Resu Chara FGFR 8/7630.pdf by guest on 20 July 2024 ing. rials and Methods nes and reagents O III, HEC-1-A, AN3 CA, SNU-16, and human lung canll lines were acquired from the American Type Culture tion (ATCC).

Techniques: Inhibition, Phospho-proteomics, Western Blot, Incubation, Software

Journal: Cancer Research

Article Title: GP369, an FGFR2-IIIb–Specific Antibody, Exhibits Potent Antitumor Activity against Human Cancers Driven by Activated FGFR2 Signaling

doi: 10.1158/0008-5472.can-10-1489

Figure Lengend Snippet: Figure 5. Effect of GP369 on SNU-16 xenografts in vivo. A, GP369 inhibits the in vivo growth of SNU-16. SCID mice inoculated with SNU-16 cells received mIgG control at 20 mg/kg or GP369 at 2, 5, 10 or 20 mg/kg by i.p. injection twice weekly. B, analysis of total and phospho-FGFR2 in SNU-16 tumors treated with either mIgG or GP369. β-Tubulin was used as a loading control. C, Phospho-RTK status of individual tumors collected at the end of study from mice treated with mIgG (top images from mouse nos. 1 and 3 in the mIgG-treated group) or GP369 (bottom images from mouse nos. 1 and 5 in the GP369-treated group). Each RTK array was probed with 250 μg of protein lysate from an individual tumor.

Article Snippet: These findings rt the hypothesis that dysregulated FGFR2 signaling is the many critical tumorigenesis pathways in human s. Targeted therapy using FGFR2 mAb could be benefir patients with aberrantly activated/amplified FGFR2 female (Taco in 1:1 Tumo calipe formu appro of 10 contro the M mice 0.72 m Resea MFMappro 10 ani by i.p. using Resu Chara FGFR 8/7630.pdf by guest on 20 July 2024 ing. rials and Methods nes and reagents O III, HEC-1-A, AN3 CA, SNU-16, and human lung canll lines were acquired from the American Type Culture tion (ATCC).

Techniques: In Vivo, Control, Injection

Journal: Cancer Research

Article Title: GP369, an FGFR2-IIIb–Specific Antibody, Exhibits Potent Antitumor Activity against Human Cancers Driven by Activated FGFR2 Signaling

doi: 10.1158/0008-5472.can-10-1489

Figure Lengend Snippet: Figure 6. Effect of GP369 on MFM-223 xenografts in vivo. A, treatment of MFM-223 xenografts with GP369 results in tumor stasis. Nude mice bearing MFM-223 cells received either mIgG or GP369 at 20 mg/kg by i.p. injection twice weekly. B, analysis of total and phospho-FGFR2 in MFM-223 tumors treated with either the control IgG or GP369. β-Tubulin was used as a loading control.

Article Snippet: These findings rt the hypothesis that dysregulated FGFR2 signaling is the many critical tumorigenesis pathways in human s. Targeted therapy using FGFR2 mAb could be benefir patients with aberrantly activated/amplified FGFR2 female (Taco in 1:1 Tumo calipe formu appro of 10 contro the M mice 0.72 m Resea MFMappro 10 ani by i.p. using Resu Chara FGFR 8/7630.pdf by guest on 20 July 2024 ing. rials and Methods nes and reagents O III, HEC-1-A, AN3 CA, SNU-16, and human lung canll lines were acquired from the American Type Culture tion (ATCC).

Techniques: In Vivo, Injection, Control