S by centrifuging at 10000 rpm for 20 min in 4uC. The protein concentration was analyzed by Bradford protein assay (Bio-Rad, USA). The entire protein was separated with ten SDS-PAGE and then transferred to a PVDF membrane (0.45 mm) for two h. Just after 2 h of blocking by five milk in TBST, incubated the membrane with mouse anti-HIF-1a (Santa Cruz, CA, USA) at 1:200 dilution and mouse anti-b-actin (MCP-1/CCL2 Protein Source proteintech, USA) at 1:2000 dilution in 4uC for 12 h and followed by two h incubating with goat anti-mouse IgG (proteintech, USA) at 1:2000 dilution. Following washing by TBST, detected the membrane signals using enhanced chemiluminescence ECL (Beyotime, China). The Image J application was applied for quantitative analysis of HIF-1a signal intensities with normalized with b-actin levels. Information had been analyzed with GraphPad Prism Version 5.0, differences among groups were statistically evalu-Analysis of differentially expressed genes in cancer versus regular tissuesGeneChip Operating Software was applied to analyze the chips and extract the raw pictures signal information. The GEO DataSets of NCBI accession number of our study is: GSE56807. Raw signal information were then imported and analyzed with Limma algorithm to recognize the differentially expressed genes. The linear models and empirical Bayes techniques have been to analyze the data. This prevented a gene with a extremely tiny fold alter from becoming judged as differentially expressed simply because of an accidentally small residual SD. The resulting P values were adjusted making use of the BH FDR algorithm. Genes had been thought of to become considerably differentially expressed if both the FDR values was ,0.05(controlling the expected FDR to no much more than five ) and gene expression showed at the least 2-fold adjustments between cancer andTable 1. GENETIC_ASSOCIATION_DB_DISEASE_CLASS analysis of 82 genes in TF-gene regulatory network.Term CancerP-Value 2.53E-Fold enrichment 2.Benjamini 4.55E-Genes TLR2, RRM2B, MDK, MMP1, TIMP1, TAP1, SERPINA1, FAS, FCGR3A, FN1, HLA-A, IGF1, CFTR, HLA-C, HLA-B, HGF, SOD1, BRCA1, CDKN1B, TFRC, PLA2G2A, IRF1, PCNA, MDM2, COL1A1, CTSB, PGK1, PARP1, GSTP1 TLR2, HLA-A, CFTR, HLA-C, OAS2, HLA-B, STAT1, MMP1, PSMB9, IFNAR2, TFRC, TAP1, IRF1, JAK1, FAS,SERPINA1, FCGR3A, GSTP1 TLR2, MMP1, TIMP1, TAP1, SERPINA3, SERPINA1, FAS, FN1,HSPA4, MYB, FCGR3A, HLA-A, IGF1, HLA-C, CFTR, HGF, HLA-B, STAT3, PSMB9, CDKN1B, PLA2G2A, COL1A2, MDM2, COL1A1, GSTP1 TLR2, OAS2, MMP1, TIMP1, CXCL10, TAP1, SERPINA3, SERPINA1, FAS, FCGR3A, HLA-A, IGF1, CFTR, HLA-C, HLA-B, STAT3, PSMB9, IFNAR2, CYBB, CD86, CTSB, IRF1, TNFRSF10B, COL1A1, PARP1, GSTPInfection Cardiovascular4.82E-06 four.77E-3.59 two.4.34E-05 2.15E-Immune2.13E-1.7.66E-doi:ten.1371/journal.pone.0099835.tPLOS One particular | plosone.orgHIF-1a and Gastric CancerFigure 3. TF-gene network of these 82 differentially expressed genes in gastric cancer tissues. Red circles in a are up-regulated genes, whereas green circles are down-regulated genes plus the yellow triangles are these five NOTCH1, Human (HEK293, His-Avi) essential TFs. B, The short framework of this network. The circles would be the clustered genes and the variety of genes is shown inside. The direction from the arrow is in the Source towards the Target. doi:10.1371/journal.pone.0099835.gated by sample one-tailed Student’s t-test with p value ,0.05 thought of as important.Construction of transcription element gene network determined by gene expression profile and transcriptional regulatory element databaseTranscription aspect (TF) gene network was constructed depending on gene expression profile and transcriptional r.