Gorization of network nodes was obtained by concentric node degree applying Concentric Measurements software50. We classified network nodes as VIPs, hubs or high-hubs by projecting all node values in a k0 (node degree) vs k1 (very first level concentric node degree) graphic.GCNs for differentially expressed GO annotated genes (DE): visualization, evaluation and neighborhood detection. Gene co-expression networks for differentially expressed GO annotated genes (DE net-Connectivity. The network connectivity k for non-directed networks was calculated by k = 2 L/N, exactly where L stands for the number of edges and N for the number of nodes10.SCIentIFIC REPORTS (2018) eight:13169 DOI:10.1038/s41598-018-31583-www.nature.com/scientificreports/Node colour yellowGene MCM2 MCM6 SMC3 CHD6 LMNB1 MCM5 NASP SMC1A TRIM28 MSH2 MSH6 XPOT RANBP9 IPO7 KPNB1 NUP93 RANBP2 XPO1 C1QBP PABPC1 DDX17 DDX5 EFTUD2 GEMIN5 SNRPB SNRPD3 POLR2A MYBBP1A RUVBL2 NOP56 PARP1 PCNA POLR2BFunction Chromatin binding/structureMM 0.72 0.87 0.61 0.53 0.24 0.23 0.16 0.24 0.MF 0.41 0.04 0.13 0.37 0.08 0.39 0.19 0.66 0.09 0.27 0.11 0.03 0.18 0.20 0.43 0.10 0.03 0.23 0.55 0.17 0.38 0.44 0.28 0.39 0.36 0.41 0.03 0.30 0.02 0.15 0.55 0.32 0.42 0.05 0.25 0.NM 0.14 0.04 0.34 0.24 0.27 0.21 0.16 0.11 0.21 0.21 0.06 0.19 0.52 0.32 0.50 0.20 NE 0.71 0.43 0.12 0.56 0.79 0.74 0.22 0.16 0.59 NE 0.29 0.25 0.38 0.01 0.38 0.67 0.17 0.29 0.NF 0.04 0.41 0.15 0.44 0.22 0.04 0.30 0.18 0.37 0.72 0.31 0.24 0.78 0.58 0.03 0.46 NE 0.25 0.34 0.21 0.32 0.11 0.35 0.13 0.72 0.54 NE 0.00 0.64 0.66 0.39 0.85 0.16 0.07 0.65 0.DNA repair Nuclear transportred blue0.49 0.22 0.81 0.67 0.30 0.42 0.63 0.63 0.Pre-mRNA processingpink0.83 0.80 0.46 0.49 0.46 0.ten 0.29 0.Transcriptiongreen0.91 0.44 0.44 0.56 0.65 0.23 0.20 0.69 0.PRKDC (DNA-PK) TOP2A CAND1 Ubiquitination brown0.Table 2. AIRE interactors’ gene-gene relationships. Pearson’s correlation coefficients for MM, MF, NM, and NF groups. Genes are classified in accordance with their molecular function. Genes presenting Pearson correlation coefficient 0.70 at the least in a single group (values in bold); NE: non-expressed.Community detection. Neighborhood detection was accomplished for DE networks applying the system proposed by Blondel et al.51, which attains great modularity values and presents excellent performance, as previously described6. Coarse-grained community structure. As a complementary evaluation for the neighborhood detection, eachGCN was rearranged in a new network accounting only for the relationships Tetraphenylporphyrin manufacturer between every neighborhood, also referred to as coarse-grained community structure (CGCS)6,52.AIRE quantification by qPCR.Reverse transcription was performed from 1 g of total RNA applying the SuperScriptTM III First-Strand Synthesis SuperMIx (Invitrogen, Carlsbad, USA). The reaction was primed with oligo DT primers and Quick SYBR Green Master Mix inside a total volume of 20 L. qPCR was performed within the StepOnePlus Real-Time PCR Method (Applied Biosystems, Foster City, USA). The typical curve method53 was used to analyze AIRE gene expression. GAPDH was used as endogenous control (see Supplementary Methods on the web). The PCR primers for AIRE have been as follows: sense 5-GGATGACACTGCCAGTCACG-3 and anti-sense 5-TCATCAGAGCTGCATGTCCC-3; and for GAPDH: sense 5-ACCACAGTCCATGCCATCAC-3 and anti-sense 5-TCCACCACCCTGTTGCTGTA-3.TM?SCIentIFIC REPORTS (2018) eight:13169 DOI:10.1038/s41598-018-31583-www.nature.com/scientificreports/ AIRE-gene expression relationships. Firstly, we Smoke Inhibitors medchemexpress chosen a set of genes coding for the Aire-targeted protei.