br RNA interference RNAi and ectopic expression To address t
RNA interference (RNAi) and ectopic expression To address the functionalities of the identified proteins, we silenced the coding genes of these proteins in a normal colonic fibroblast cell line, CCD-18Co, and then performed coculture assays with colon cancer cells. Follistatin-related protein 1 (FSTL1) was chosen for ectopic expression analysis in CCD-19Co, which was used for coculture assays. The small interference RNAs (siRNAs) and cloning primers are listed in Supplementary Table 5.
Functional enrichment analysis of the identified proteins GO enrichment analysis was performed using DAVID and significant enrichment categories were accepted with p value and Benjamini value less than 0.05. The enrichment analysis results are shown in Supplementary Table 6. Tissue specificity gene expression enrichment was analyzed using the Gene Enrichment Profiler (http://xavierlab2.mgh.harvard.edu/EnrichmentProfiler/). The results for tissue expression enrichment are embedded in Supplementary Table 2.
Comparison with the known identifications of colon cancer cells The identified fibroblast proteins were compared with a batch of known proteomic analyses of colon cancer prolyl hydroxylase inhibitor to evaluate their specific expression properties [7–16]. The identified proteins of these reports varied from ~40 to 2300. The comparison was mainly based on official gene symbols. In cases when needed, the International Protein Index (IPI) accessions were mapped to gene symbols and UniProt accessions using the Protein Identifier Cross-Reference (PICR) (http://www.ebi.ac.uk/Tools/picr/). The analyzing results were integrated in Supplementary Table 2. Area-proportional Venn diagram was generated with BioVenn (http://www.cmbi.ru.nl/cdd/biovenn/index.php).
Data, experimental design, materials and methods 3429 Non-redundant proteins discovered in our urine proteomic analysis are characterized and are noted in Supplementary Table 1. 1615 of these proteins were contained in vesicles while the remaining 1794 were equally distributed among CPLL (1488) and butanol insoluble fractions (322). Several proteins were detected exclusively in one of the phases of the procedure, suggesting that each step is crucial in the fractionation strategy. Many (1724) proteins are here described whose presence in urine have never been reported and represent a potential source of information considering that urine is the unique site of excretion of products of interaction of metabolic processes.
Conflict of interest
Acknowledgments The Giannina Gaslini Institute provided financial and logistic support to the study. This work was also supported by the Ministry of Health, Italy ‘Ricerca Corrente’ and from contributions derived from ‘Cinque per mille dell’IRPEF’. We also acknowledge contributions from the Renal Child Foundation, Fondazione La Nuova Speranza (‘Progetto integrato per la definizione dei meccanismi implicati nella glomerulo sclerosi focale’) and Italian Society of Nephrology (Progetto Ricercando).
Data are supplied here and have been deposited to the open access library of ProteomeXchange Consortium () via the PRIDE partner repository with the dataset identifier
Data, experimental design, materials and methods
Conflict of interests
Acknowledgments This work is supported by grants to NG from the French National Agency for Research (ANR-10-INTB-1301-PARACTIN) and from the French Parasitology Network of Excellence ParaFrap (Grant ANR-11-LABX0024). DP was supported by fellowships from the French Ministère de la Recherche et la Technologie (MRT) and from Fondation pour la Recherche Médicale (FRM). GDJ is supported by the Wellcome Trust–DBT India Alliance (Grant 500080/Z/09/Z).
Value of the data
Data, experimental design, materials and methods We describe here a unique dataset composed of qualitative proteomic analysis of chicken spermatozoa and seminal plasma and quantitative proteomic analyses related to semen quality. The present study identified a large number of proteins that have never previously been described in Gallus gallus. Furthermore, label free quantitative proteomic analyses combined with physiological tests allowed phenotyping semen at the individual level and characterization of new peptides and proteins that are biomarker candidates of fertility.