The fold change cutoff between control and
Nge of gene expression [45,46]. The fold change cutoff between control and exposed samples wasThe MS/MS spectra were searched against the SwissProt database (release SwissProt_2013_01.fasta) totaling 65,754 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/10811056 protein sequences from mammals. Searches for trypsic peptides were performed with the following parameters: full-trypsin specificity, a mass tolerance of 5 ppm on the parent ion and 0.5 Da on the MS/MS, carbamidomethylPisani et al. BMC Genomics (2015) 16:Page 13 ofCys as static modification and oxidized Met as dynamic modification, and maximum number of missed cleavages set at 2. All peptide matches with a peptide score below a p-value of 0.05 were filtered. A protein was considered to be validated when at least two different peptides were detected in the same experiment. The false-positive rate for protein identification was estimated using the appropriate decoy database as below 1 . The number of MS/MS spectra per protein (spectral counts) was determined for each sample. Spectral counts PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/22763976 were then compared using the TFold method of the PatternLab software [49]. Fold change and p-value cutoffs were set at 2.0 and 0.05, respectively.Integrative biological analysisfor each of the nine samples; control (Ctrl), first concentration (C1), and second concentration (C2), with three replicates, are indicated, as well as the PatternLab statistical results (fold change, p-value, signal +, signal -, class) for the three possible comparisons (C1 versus Crtl; C2 versus Crtl; C2 versus C1). The patternLab classes are: blue (identifications that satisfied both the fold and statistical criteria), orange (identifications that did not meet the fold criterion but 3-Bromo-2-methoxyaniline have low p-values), green (identifications that satisfied the fold criteria but this was most likely to have happened by chance), and red (identifications that did not meet the fold and 5(2H p-value criteria). Additional file 3: Table S3. List of genes and proteins supporting the hierarchical stress response. A549 cells were exposed to 1.5, 3.0, and 6.0 g/cm2 silica NPs for 24 h. Fold changes of genes and proteins significantly up- or downregulated were determined by an unpaired t-test (p < 0.05) and a false discovery rate correction. Gene symbols are indicated in italics, protein symbols are not italicized. * Transcripts whose expression is identical in control cells and in NPs-exposed cells. Additional file 4: Table S4. List of primers used in qRT-PCR. Differential analysis of transcripts from cells exposed to 6.0 g/cm2 SiO2 NPs for 72 h versus unexposed cells was performed by qRT-PCR with the Sybr Green PCR Master Mix (Finzyme) kit according to the manufacturer's instructions on Opticon II (Biorad). This pool of genes represents the "coagulation system" pathway. Competing interests The authors declare that they have no competing interests. Authors' contributions CP carried out the cell culture, viability studies, size determination, microarray experiments, analyzed the data, and drafted the manuscript. JCG performed the mass spectrometry experiments. VN carried out the qRT-PCR experiments. MO performed the AFM characterization. JA participated in the exoproteomics study and contributed to the manuscript writing. OP conceived the study and its design, coordinated the experiments, analyzed the data, and wrote the 2-Amino-3-methoxypyridine final manuscript. All the authors read and approved the final manuscript. Acknowledgements We thank J e Rose from CEREGE (Centre de Recherche et d'Enseignement de G sciences de l'Environn.
