Gorization of network nodes was obtained by concentric node degree using Concentric Measurements software50. We classified network nodes as VIPs, hubs or high-hubs by projecting all node values within a k0 (node degree) vs k1 (very first level concentric node degree) graphic.GCNs for differentially expressed GO annotated genes (DE): visualization, analysis and community 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, where L stands for the number of edges and N for the number of nodes10.SCIentIFIC REPORTS (2018) 8:13169 DOI:ten.1038/s41598-018-31583-www.nature.com/scientificreports/Node color 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 a minimum of in a single group (values in bold); NE: non-expressed.Community detection. Neighborhood detection was achieved for DE networks applying the strategy proposed by Blondel et al.51, which attains great modularity values and presents great overall performance, as previously described6. Coarse-grained community structure. As a complementary evaluation for the neighborhood detection, eachGCN was rearranged inside a new network accounting only for the relationships among each community, also called coarse-grained neighborhood structure (CGCS)6,52.AIRE quantification by qPCR.Reverse transcription was performed from 1 g of total RNA working with the SuperScriptTM III First-Strand Synthesis Lufenuron Anti-infection SuperMIx (Invitrogen, Carlsbad, USA). The reaction was primed with oligo DT primers and Fast SYBR Green Master Mix inside a total volume of 20 L. qPCR was performed in the StepOnePlus Alprenolol In Vitro Real-Time PCR Method (Applied Biosystems, Foster City, USA). The common curve method53 was employed to analyze AIRE gene expression. GAPDH was employed as endogenous control (see Supplementary Approaches on the web). The PCR primers for AIRE were 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:ten.1038/s41598-018-31583-www.nature.com/scientificreports/ AIRE-gene expression relationships. Firstly, we selected a set of genes coding for the Aire-targeted protei.