dataesr/scientific-paragraphs-categorization huggingface dataset:
Generating the set of antibody-antigen complexes The structures of the antibody/antigen complexes from the PDB were retrieved from the IMGT/3Dstructure-DB database (IMGT®, the international ImMunoGeneTics information system® † )  [52]. A total of 506 PDB files of immunoglobulin antibody or antibody fragments in complex either with protein or with peptide ligands were retrieved from the database (data from May 2011). We extracted 484 VH, 404 VL-κ, and 59 VL-λ sequences from these files. The difference between the number of PDB files and antibody sequences extracted is due to files containing Fc domains only (lacking variable chains), which we removed from the set. VH sequences were clustered using program cd-hit  [53]  with default parameters and a 92% identity cutoff value, and the sequence with the best resolution was kept in each cluster, resulting in 238 VH, 183 VL-κ, and 30 VL-λ.
All files were renumbered using AHo numbering scheme  [17]  that minimizes the average deviation of residues in aligned three-dimensional structures of immunoglobulin domains, using a set of already aligned variable domains present in the original publication  [17] , clustalw software, and in-house python scripts. Unconventional sequences that were incompatible with AHo numbering were discarded resulting in 234 VH, 183 VL-κ, and 30 VL-λ sequences. When multiple molecules were present in the crystal asymmetric unit, only a single copy of the variable domain was kept and renamed "H", "L", or "K" for VH, VL-λ, and VL-κ, respectively (in-house python script). Ligand interacting chains were defined as possessing at least one contact with antibody CDR residues using NCONT program from CCP4 suite  [54].
Structures devoid of any ligand chain interacting with the variable domains were discarded. Inspection of these particular files showed that either the ligands were haptens mis-annotated as peptide in the IMGT (thus ignored by NCONT) or the interaction was through the Fc domain. The final antibody-antigen database consisted of 227 VH, 175 VL-κ, and 31 VL-λ, including the structure solved in this study.
Data collection, structure determination, and refinement The proteins were expressed and purified as previously described  [31]. Crystals of the complexes of Gankyrin and scFv F5 were obtained at 17 °C by vapor diffusion in sitting drops by mixing 0. 2 μl of the protein solution and 0. 2 μl of reservoir solution containing 0. 1 M Hepes (pH 7. 0) and 8% polyethylene glycol 8000. The crystal was mounted in a fiber loop and flash-cooled in liquid nitrogen after cryoprotection with the reservoir solution plus 15% ethylene glycol. Data collection from a frozen single crystal was performed at 100 K on the beamline ID29 of the European Synchrotron Radiation Facility (Grenoble, France). The crystal belongs to the trigonal space group P3 2 21, with one Gankyrin-scFv F5 complex per asymmetric unit. The data were integrated and scaled using HKL2000  [55]  (see statistics in Table  1 ).
The structures were solved by molecular replacement using the program AMoRe  [56]. The structure of Gankyrin and a neutralizing antibody F10 [PDB ID: 1UOH and PDB ID: 3FKU (chain X), respectively] were used as starting models  [34, 57]. Refinement involved iterative cycles of manual building and refinement calculations. The programs phenix. refine [58]  and Coot  [59]  were used throughout structure determination and refinement. Several terminal residues and 16 residues (119-134) of the linker of the scFv F5 are not modeled as electron density map was poor in the corresponding regions. Anisotropic scaling, a bulk solvent correction, and TLS restrains were used. Five TLS groups for Gankyrin and four groups for scFv F5 were generated by using the program TLSMD  [60]. Individual B atomic factors were refined isotropically.
Solvent molecules were then placed according to unassigned peaks in the electron density map. In the Gankyrin-scFv F5 complex, refined at 2. 5 Å with no σ cutoff, the final model contains 224 residues  for Gankyrin, 225 residues (3-119 and 134-244) for antibody scFv F5, and 428 water molecules. Free R value was calculated as described previously  [61]. Structural figures were generated by using the program PyMOL  [62].
Acknowledgments This work and the salary of G. R. were supported by the  SELAS Docteur l'Hermite Collaboration  Agreement No. 10561A10. Authors thank  Labex MabImprove  for its continuous support ( French National Research Agency  under the program " Investissements d'Avenir "  Grant Agreement LabEx MAbImprove :  ANR-10-LABX-53 ),  François Stricher  for his help with FoldX, and  Pierre Poussin-Courmontagne  ( Structural Biology and Genomics Platform, Institut Génétique Biologie Moléculaire Cellulaire ) for help in crystallization. N. R. thanks support by institutional funds from the  Centre National de la Recherche Scientifique , the  Institut National de la Santé et de la Recherche Médicale , the  Université de Strasbourg, Instruct , part of the  European Strategy Forum on Research Infrastructures , and by national member subscriptions, the  French Infrastructure for Integrated Structural Biology.
Catalase Assay. Catalase activity in the colon was determined by the method of Aebi  [39]. The principle of the assay is based on the determination of the rate constant of hydrogen peroxide decomposition by the catalase enzyme. Decomposition of H 2 O 2 was followed directly by monitoring the decrease of absorbance at 240 nm. Data were expressed as unit per mg -1 protein. 2. 12. Reduced Glutathione Content. Glutathione (GSH) level in the colon was measured by the method of Ellman  [40]. The GSH levels were estimated by the reduction of DTNB (dithiobis-2-nitrobenzoic acid). Tissue homogenates were deproteinized and centrifuged at 600 g for 20 min. After addition of DTNB and phosphate buffer (pH 8. 0) into the cleared sample supernatants, the yellow color developed was read at 412 nm. Results were expressed as nmol of GSH mg -1 protein (molar extinction coefficient: 1.
The fact that apocynin inhibits mRNA and protein expression of NOX1 subunits suggests that ROS could regulate the expression of these subunits in a positive control feedback loop. Taking into account the positive effects of apocynin on all these activation was assessed by Western blot using phospho-ERK1/2 specific antibodies in colon of mice treated with TNF (10 g ⋅ kg -1 , ip) in the presence or absence of apocynin (25 mg ⋅ kg -1 , ip) (C in the figure is for control untreated mice). Total ERK1/2 protein, assessed with an ERK1/2 antibody, was used as loading control. Lower: densitometric analysis of the ratio of phospho-ERK1/2 to total ERK1/2 protein. Data are expressed as means ± S. E. M from  = 5 mice in each group. *  < 0. 05, * *  < 0. 01, and * * *  < 0. 001 versus control; #  < 0. 05, ##  < 0.
01, and ###  < 0. 001 versus TNF. (b) Upper: p38MAPK activation was assessed by Western blot using phospho-p38MAPK specific antibodies in colon of mice treated with TNF (10 g ⋅ kg -1 , ip) in the presence or absence of apocynin (25 mg ⋅ kg -1 , ip) (C in the figure is for control untreated mice). Total p38MAPK protein, assessed with a p38MAPK antibody, was used as loading control. Lower: densitometric analysis of the ratio of phospho-p38MAPK to total p38MAPK protein. Data are expressed as means ± S. E. M from  = 5 mice in each group. *  < 0. 05, * *  < 0. 01, and * * *  < 0. 001 versus control; #  < 0. 05, ##  < 0. 01, and ###  < 0. 001 versus TNF. (c) Upper: JNK activation was assessed by Western blot using phospho-JNK specific antibodies in colon of mice treated with TNF (10 g ⋅ kg -1 , ip) in the presence or absence of apocynin (25 mg ⋅ kg -1 , ip) (C in the figure is for control untreated mice).
Total JNK protein, assessed with a JNK antibody, was used as loading control. Lower: densitometry analysis of the ratio of phospho-JNK to total JNK. Data are expressed as means ± S. E. M from  = 5 mice in each group. *  < 0. 05, * *  < 0. 01, and * * *  < 0. 001 versus control; #  < 0. 05, ##  < 0. 01, and ###  < 0. 001 versus TNF. parameters and its very low toxicity  [28] , this component may be useful for the treatment of IBD. However, TNF which is a dominant player in the pathogenesis of IBD  [54]  induces an acute process of inflammation, which probably only mimics the active phase of colitis. Therefore, effectiveness of apocynin might be limited to the acute phase of the disease. We plan in future experiments to investigate if apocynin has a protective effect on a chronic animal model of the disease such as chronic model based on repeated administration of 2,4,6trinitrobenzene sulfonic acid (TNBS).
Figure8: Redox-sensitive MAP kinases are activated in the colon of TNF-treated mice: modulation by apocynin. (a) Upper: ERK1/2 activation was assessed by Western blot using phospho-ERK1/2 specific antibodies in colon of mice treated with TNF (10 g ⋅ kg -1 , ip) in the presence or absence of apocynin (25 mg ⋅ kg -1 , ip) (C in the figure is for control untreated mice). Total ERK1/2 protein, assessed with an ERK1/2 antibody, was used as loading control. Lower: densitometric analysis of the ratio of phospho-ERK1/2 to total ERK1/2 protein. Data are expressed as means ± S. E. M from  = 5 mice in each group. *  < 0. 05, * *  < 0. 01, and * * *  < 0. 001 versus control; #  < 0. 05, ##  < 0. 01, and ###  < 0. 001 versus TNF. (b) Upper: p38MAPK activation was assessed by Western blot using phospho-p38MAPK specific antibodies in colon of mice treated with TNF (10 g ⋅ kg -1 , ip) in the presence or absence of apocynin (25 mg ⋅ kg -1 , ip) (C in the figure is for control untreated mice).
Total p38MAPK protein, assessed with a p38MAPK antibody, was used as loading control. Lower: densitometric analysis of the ratio of phospho-p38MAPK to total p38MAPK protein. Data are expressed as means ± S. E. M from  = 5 mice in each group. *  < 0. 05, * *  < 0. 01, and * * *  < 0. 001 versus control; #  < 0. 05, ##  < 0. 01, and ###  < 0. 001 versus TNF. (c) Upper: JNK activation was assessed by Western blot using phospho-JNK specific antibodies in colon of mice treated with TNF (10 g ⋅ kg -1 , ip) in the presence or absence of apocynin (25 mg ⋅ kg -1 , ip) (C in the figure is for control untreated mice). Total JNK protein, assessed with a JNK antibody, was used as loading control. Lower: densitometry analysis of the ratio of phospho-JNK to total JNK. Data are expressed as means ± S. E. M from  = 5 mice in each group.
2 ERK 1 / 2 #Figure 8 : 2128 Figure8: Redox-sensitive MAP kinases are activated in the colon of TNF-treated mice: modulation by apocynin. (a) Upper: ERK1/2 activation was assessed by Western blot using phospho-ERK1/2 specific antibodies in colon of mice treated with TNF (10 g ⋅ kg -1 , ip) in the presence or absence of apocynin (25 mg ⋅ kg -1 , ip) (C in the figure is for control untreated mice). Total ERK1/2 protein, assessed with an ERK1/2 antibody, was used as loading control. Lower: densitometric analysis of the ratio of phospho-ERK1/2 to total ERK1/2 protein. Data are expressed as means ± S. E. M from  = 5 mice in each group. *  < 0. 05, * *  < 0. 01, and * * *  < 0. 001 versus control; #  < 0. 05, ##  < 0. 01, and ###  < 0. 001 versus TNF. (b) Upper: p38MAPK activation was assessed by Western blot using phospho-p38MAPK specific antibodies in colon of mice treated with TNF (10 g ⋅ kg -1 , ip) in the presence or absence of apocynin (25 mg ⋅ kg -1 , ip) (C in the figure is for control untreated mice).
Data analyses were performed using the LightCycler software (Roche) and normalized with respect to invariant S26 mRNA levels. The sequences of the primers used for amplification are given in Supplementary Data S1. Semi-quantitative PCRs were performed with the Go Taq polymerase from Promega (M500B) using 30 amplification cycles and analyzed by electrophoresis through 1% agarose gels.
Statistical analyses Data were reported as means ± standard deviations. For calculations of means in ChIPs, the values of the signals at position -1. 9 were arbitrarily referred as 1, except for H3K4me 3 and H3K9ac, where the signal position at -1. 3 was set to 1, as well as for Pol II, P-S2 and H3K36me 3 , where the signals at position 3. 5 were set to 1. For calculation of means in RT-qPCR, the values of the signals for control conditions were set to 1. Significance between two conditions was assessed by the Student's paired t-test using the GraphPad Prism 5 software. P-values were considered as significant when *≤0. 05 and highly significant when **≤0. 01, ***≤0. 0005 or ****≤0. 0001.
Binding of Fra-1 to the Plau promoter region Bioinformatic analyses revealed the presence of at least 40 potential AP-1-binding sites (see Supplementary Data S2) at the Plau locus in the -6. 5/+6. 3 kb region (nucleotides are numbered herein with respect to the Plau-001 transcription start site (TSS), as positioned in the Ensembl database (http://www. ensembl. org/Homo sapiens/ Info/Index). Among them, (i) an enhancer with 2 AP-1responsive sites has already been identified in human liver cells at -1. 9 kb (8-10), (ii) an AP-1-binding site was described in mouse NIH 3T3 fibroblasts at -6. 9 kb (6) in a domain homologous to the human gene -5. 3 kb region and (iii) publicly available data (http://genome. ucsc. encode/) show AP-1 binding at -4. 2 kb in human hepatocyte HepG2 cells. To assess which sites in the -6.
Transcriptional complexity upstream of the Plau-001 TSS An intriguing question was the role/activity of Pol II upstream of the Plau-001 TSS, which could be synthesis of sense and/or antisense RNA species and/or abortive transcription or Pol II tracking toward the TSS. To address this, we first assessed the expression of a RNA transcript, called Plau-004, described in the Ensembl database (http://www. ensembl. org/Homo sapiens/ Gene/Summary?g=ENSG00000122861;r=10:75668935-75677255) without any indications on its function, its overall structure and/or its expression territories/conditions. The available sequence data indicate that it starts at -1. 955 kb upstream of the Plau-001 TSS and that its short first exon is connected to exons 2, 3, 4 and at least part of exon 5 of Plau-001 (Figure  4A ).
Transcription of Plau-001 Our data indicate that Fra-1 is recruited at two AP-1 binding sites showing typical additional enhancer signatures  (41) , i. e. low nucleosome density and binding of p300. One enhancer (ABR-1. 9) is located 1. 9 kb upstream of Plau-001 TSS, as already described in other cell contexts  (8) (9) (10). The other one (ABR-4. 1) is located 4. 1 kb upstream of Plau-001 TSS and has been reported in a publicly available database (http://genome. ucsc. encode/) to be an AP-1-binding site in human hepatocytic HepG2 cells. However, in its present state, our study does not exclude that other AP-1-binding sites located further upstream may also participate to uPA (de)regulation. Strikingly, the distribution of Pol II (as well as that of Mediator) on the Plau locus strongly suggests that Pol II is not recruited at the level of Plau-001 TSS in MDA-MB231 cells but rather in the TSS upstream region up to ABR-4.
9 and Plau-001 TSS is reminiscent of the abovementioned cases of Pol II tracking  (55) (56) (57) (58) , although the fraction of Pol II actively transcribing during tracking has not been addressed in the latter situations. It is therefore of note that nascent RNAs are of rather equivalent abundance upstream and downstream of Plau-001 TSS. This implies that most, if not all, Pol II molecules that transcribe Plau-001 have most likely previously tracked between the enhancer and the TSS. Interestingly, data obtained in the K562 cell line and available in the UCSC databank reveal low amounts of PolyA + nuclear RNA (>200 bp) transcribed in the -1. 5 kb/TSS region, suggesting that tracking during uPA gene expression may not be restricted to MDA-MB231 cells. It is also worth considering that tracking Pol II molecules are essentially phosphorylated on CTD Ser5, whereas Plau-001-generating ones are phosphorylated on Ser2.
Transcription of Plau-004 Plau-004 RNA is detectable in MDA-MB231 cells, as well as in other aggressive breast cancer cell lines. It is, however, expressed at very low levels, not only in comparison to Plau-001 in total, but also in comparison to nascent RNAs in the region where Pol II tracks before reaching Plau-001 TSS. Its initiation site is located in the ABR-1. 9 region with a first exon spanning nucleotides -1980 to -1862. Moreover, RT-PCR analyses indicate that its description found in the Ensembl database is incomplete (reported end in exon 5 of Plau-001) and that, most probably, Plau-004 ends like Plau-001. An important question will now be to identify its biological function(s). Interestingly, our data indicate that Fra-1 operates as a transcriptional activator for Plau-001 but has a negative effect on Plau-004 synthesis in MDA-MB231 cells.
FUNDING MP's laboratory is funded as an "Equipe Labellisée" of the French Ligue Nationale contre le Cancer. This work was also supported by the CNRS, the "Programme Blanc" of the Agence Nationale pour la Recherche (ANR) and the regional Languedoc-Roussillon Ligue contre le Cancer. G. M-T was supported by fellowships from the Ministry of Higher Education and Research (MESR) and from the Association pour la Recherche contre le Cancer (ARC). C. T. is supported by a fellowship from The Ligue Nationale contre le Cancer. The open access charge is funded by the Ligue Nationale contre le Cancer. Conflict of interest statement. None declared.
The Ki67 value was determined by immunohistochemistry. The LOH status of 17p13 was inferred from the study of microsatellite markers  [27]. Molecular markers of risk of recurrence and poor prognosis were described previously from quantitative RT-PCR measurements of DLGAP5 (DLG7), PINK1 and BUB1B expression  [21]. Informed signed consent for the analysis of the tumor and for access to the data collected was obtained from all the patients, and the study was approved by the institutional review board of the Cochin Hospital. Cellular models of IGF2 knock-down in H295R cells H295R cells, which are derived from an adrenal carcinoma overexpressing IGF2, were ordered from ATCC, and cultured as described previously  [28]. For the transient knock-down of IGF2, siRNA duplexes against IGF2 (forward sequence 59UCGUUGAGGAGUGCUGUU-UdTdT39) and a control siRNA with no target in the human genome (forward sequence 59GGCAUAGAUGUAGCUGUA-AdTdT39) were designed and obtained from Eurogentec.
Cell Proliferation, apoptosis and cell cycle assays Cell proliferation was measured with the CellTiter 96 wells Non-Radioactive Cell Proliferation Assay (Promega), according to manufacturer's instructions (quick protocol). Cells (8000/well) were plated on 96-well plates and the first measurement (D0) was made 3 days later. Optical absorbance was measured at 570 nm in a microplate reader. Apoptosis was analyzed by staining cells with Annexin V with the Annexin-V-FLUOS Staining Kit (Roche), according to manufacturer's instructions. Cells were analyzed by flow cytometry with a Cytomics TM FC500 (Beckman Coulter), and data were processed with Cytomics RXP software. Spontaneous apoptosis was studied in stable clones after 10 days of IGF2 knock-down induced by doxycycline treatment. TNF-alpha-induced apoptosis was analyzed in H295R cells after transient IGF2 knock-down by siRNA for 48 h, followed by a 48 h treatment with 20 ng/mL TNF-alpha (eBioscience).
For cell cycle analysis, cells were seeded in 6-well plates. At time of analysis, cells were trypsinized, rinsed with PBS and fixed in 500 mL of 70% ethanol. They were then centrifuged, rinsed in PBS, and suspended in 50 mg/mL of propidium iodide + 100 mg/ mL RNase A. Analysis by flow cytometry was performed with a Cytomics TM FC500 (Beckman Coulter), and data were processed with Cytomics RXP software.
Briefly, 2. 5 ml of 10-fold diluted reverse transcription product was used with 500 nM of each primer in a 10 ml reaction. Optimal annealing temperature, PCR efficiency, and PCR specificity (dissociation curve to verify amplification of one product) were determined with a four-point dilution curve. Conditions are described for all target genes in Table  S1. Analyses for tumors. Relative quantification of target cDNA was determined by calculating the difference in crossthreshold (C T ) values after normalization to 18SRNA5 signals (DDC T method), taking a normal adrenal sample as a reference. In our previous study, 18S mRNA was validated as the best reference gene, with perfect correlation between microarray and qPCR results  [21]. Similar results were obtained using GAPDH as reference gene (data not shown).
Western blot IGF2-high and IGF2-low ACC (10 samples each) were studied by western blot. Whole cell protein lysates were obtained from tumors by extraction with RIPA lysis buffer (50 mM Tris-HCl, pH 7. 5, 1 mM EDTA, 150 mM NaCl, 0. 1% v/v Nonidet P-40 (NP40), 1 mM dithiothreitol (DTT)) with inhibitors of proteases (Complete Protease Inhibitor Cocktail Tablets, Roche) and phosphatases (PhosSTOP Phosphatase Inhibitor Cocktail Tablets, Roche). Western blotting was done according to standard procedures  [30]  with chemiluminescence and films (Amersham hyperfilm ECL, GE healthcare). Antibodies and conditions are described in Table  S2. Western blot bands were quantified with the GeneTools software (http://www. syngene. com/genetools-software-download).
Transcriptomic analyses Transcriptomic analyses of adrenocortical tumors (47 ACA, 23 IGF2-high ACC and six IGF2-low ACC) were reported previously  [21]. All data are available at the ArrayExpress Web site (http:// www. ebi. ac. uk/arrayexpress, experiment E-TABM-311) and were confirmed by quantitative RT-PCR of more than 100 genes. Transcriptomic analyses of three different H295R clones, with or without IGF2 knock-down for 2 or 10 days, was carried with Human gene 1. 0 ST array (Affymetrix). Moreover, the same analysis was done after a 5 day transient inhibition of IGF2 by siRNA in H295R cells, in normal (2% of Ultroser G serum, Pall Biosepra) or depleted medium (without UltroserG, depletion from one day before siRNA transfection to the end of the experiment). Total RNA was extracted from cell lines with the RNeasy Mini kit and RNase-free DNase Set (Qiagen) according to the manufacturer's instructions, and RNA quality was evaluated by Bioanalyzer (Agilent).
Statistical analyses Survival Kaplan-Meier curves, Fisher and Wilcoxon statistical tests were carried out with R software. Genes differentially expressed between IGF2-high and IGF2low ACCs in the transcriptomic study were determined with the limma t-test (R package). Genes that showed significantly modified expression following IGF2 knock-down in cellular models were defined with a paired ttest on the eight independent cellular experiments (three clones analyzed at 2 and 10 days after IGF2 knock-down, and two siRNA experiments with 5 days of IGF2 knock-down in normal or depleted medium, see above). Pathways analysis was performed as follows: (i) Pathways were obtained from KEGG, Gene Ontology, Biocarta, MSigDB and Stanford Microarray Database websites. (ii) Gene lists tested for enrichment were based on the top 500, 250 and 100 probe sets sorted by p-value (for a given comparison -paired t-test in cell models and limma t-tests in tumors).
Expression and activity of IGF signaling pathways in IGF2high and IGF2-low tumors and cells Data from microarray experiments analyzing the transcriptome of adrenocortical tumors  [21]  suggest few differences in the expression of IGF pathway members between IGF2-low and IGF2-high carcinoma. These results were obtained with a limited number of IGF2-low ACC (n = 6); therefore, we sought to validate these findings. We assessed the expression of 18 partners associated with the IGF signaling pathway, including receptors, IGFBPs, Akt, PI3K and Erk isoforms, by quantitative RT-PCR in 10 IGF2-low and the 23 IGF2-high carcinoma. In addition, we analyzed the protein abundance of receptors, Akt and Erk isoforms by western blotting in 10 tumors of each group. Most of the 18 studied genes were not differentially expressed between IGF2-high and IGF2-low ACC, and the few significant differences were modest (Figure  2A ).