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1 1 ONLINE SUPPLEMENT 2 TITLE PAGE 3 4 Original Article 5 6 dsRNA-induced changes in gene expression profiles of primary 7 nasal and bronchial epithelial cells from patients with asthma, 8 rhinitis and controls 9 10 Ariane H Wagener, MD,a Aeilko H Zwinderman, PhD,b Silvia Luiten,c Wytske J Fokkens, 11 MD, PhD,c Elisabeth H Bel, MD, PhD,a Peter J Sterk, MD, PhD,a and Cornelis M van Drunen, 12 PhD,c 13 14 a 15 The Netherlands 16 b 17 Center, University of Amsterdam, The Netherlands 18 c 19 The Netherlands Department of Respiratory Medicine, Academic Medical Center, University of Amsterdam, Department of Clinical Epidemiology, Biostatistics & Bioinformatics, Academic Medical Department of Otorhinolaryngology, Academic Medical Center, University of Amsterdam, 20 21 Corresponding author 22 Ariane H. Wagener, MD 23 Department of Respiratory Medicine, F5-260 24 Academic Medical Center (AMC) 2 25 University of Amsterdam 26 Meibergdreef 9 27 1105 AZ Amsterdam 28 The Netherlands 29 Telephone: +31 (0)20 5661660 30 E-mail: [email protected] 3 31 METHODS 32 Primary epithelial cell culture 33 Epithelal cell cultures were done as previously described [1]. Primary cells were obtained by 34 first digesting the biopsies and brushes with collagenase 4 (Worthington Biochemical Corp., 35 Lakewood, NJ, USA) for 1 hour in Hanks’ balanced salt solution (Sigma-Aldrich, 36 Zwijndrecht, The Netherlands). Subsequently cells were washed with Hanks’ balanced salt 37 solution (HBSS) and resuspended in BEGM (Invitrogen, Breda, The Netherlands) and seeded 38 in one well of a 6 wells plate. Cells were grown in fully humidified air containing 5% CO2 at 39 37°C, and culture medium was replaced every other day. Cells were cultured to 80% 40 confluence and were pre-incubated with BEBM for 24 hours prior to exposure to BEBM 41 containing 20µg/ml poly(I:C) or with BEBM alone (control condition) for 24 hours before 42 removal of supernatant and RNA extraction. For bronchial epithelial cells it took 14 days on 43 average, and for nasal epithelial cells it took 24 days on average to grow to 80% confluence. 44 There was no difference in time of culture between the three subject groups. 45 46 RNA extraction 47 Total RNA from each sample was extracted using Trizol (Life Technologies Inc., Gaitersburg, 48 MD, USA) using manufacturer’s protocol, followed by purification by nucleospin RNA II 49 (Machery-Nagel, Düren, Germany). RNA concentration of all samples was measured on the 50 nanodrop ND-1000 (NanoDrop Technologies Inc., Wilmington, DE, USA). The quality of the 51 RNA was checked by using Agilent 2100 bio-analyser (Agilent Technologies, Palo Alto, CA, 52 USA). All RIN scores were ≥9.5. 53 54 Microarray Affymetrix U133+ PM 4 55 Microarray analysis was done by previously published method [1]. Human Genome U133+ 56 PM Genechip Array (Affymetrix inc., Santa Clara, CA, USA) representing more than 47,000 57 transcripts and variants, including over 33,000 well-characterized genes, was used in the 58 analysis of the genes. The MicroArray Department (MAD) of the University of Amsterdam, a 59 fully licensed microarray technologies centre for Affymetrix Genechip® platforms, 60 performed the technical handling and the quality control of the microarray experiments. The 61 quality of the images was checked by visual inspection and all raw data passed quality 62 criteria based on borderplots, pseudocolor slide images, RNA degradation plots, box and 63 density plots, RI plots (against a pseudoreference), correlation and PCA plots. 64 65 Real-time polymerase chain reaction and analysis 66 Quantitative real-time PCR was used to validate the differential expression of selected genes. 67 A random selection of 9 genes was used that showed a variance of responses to Poly(I:C). 68 PCR was performed on Bio-Rad CFX96 real-time PCR detection system (Bio-Rad, 69 Veenendaal, The Netherlands). SYBR® Green primer sequences for IL13-Rα2, EREG, 70 PDE4D, IL1-β, IP-10, TIMP2, IL8, β –actin and GAPDH were obtained from Sigma-Aldrich 71 (Sigma-Aldrich, Zwijndrecht, The Netherlands). The following primers were used: IL13-Rα2; 72 sense: TGC-TCA-GAT-GAC-GGA-ATT-TGG, antisense: TGG-TAG-CCA-GAA-ACG- 73 TAG-CAA-AG, EREG; sense: ATC-CTG-GCA-TGT-GCT-AGG-GT, antisense: GTG-CTC- 74 CAG-AGG-TCA-GCC-AT, 75 antisense: ACC-AGA-CAA-CTC-TGC-TAT-TCT, IL1-β; sense: GGA-TAT-GGA-GCA- 76 ACA-AGT-GG, antisense: ATG-TAC-CAG-TTG-GGG-AAC-TG, IP-10; sense: TGA-AAT- 77 TAT-TCC-TGC-AAG-CCA-AT, antisense: CAG-ACA-TCT-CTT-CTC-ACC-CTT-CTT-T, 78 TIMP2; sense: ATA-AGC-AGG-CCT-CCA-ACG-C, antisense: GAG-CTG-GAC-CAG- 79 TCG-AAA-CC, IL8; PDE4D; sense: sense: GGC-CTC-CAA-CAA-GTT-TAA-AA, CCA-CAC-TGC-GCC-AAC-ACA-GAA-ATT-ATT-G, 5 80 antisense: GCC-CTC-TTC-AAA-AAC-TTC-TCC-ACA-ACC-C, β –actin; sense: TGA-GCG- 81 CGG-CTA-CAG-CTT, antisense: TCC-TTA-ATG-TCA-CGC-ACG-ATT-T, GAPDH; sense: 82 GAA-GGT-GAA-GGT-CGG-AGT-C, antisense: 83 For ATF3 and DUSP1 we used TaqMan® gene expression assays from Applied Biosystems 84 (Nieuwerkerk a/d IJssel, The Netherlands) with the following assay IDs: ATF3; 85 HS00231069_M1, DUSP1; HS006102757_G1. 86 Correlations between fold changes (FC) within the microarray data and the real-time PCR 87 data were determined using Pearson’s correlation. GAA-GAT-GGT-GAT-GGG-ATT-TC. 6 88 RESULTS 89 Validation of microarray data 90 The results of this microarray experiment were validated by independent real time PCR on the 91 same starting material used for the microarray analysis. We first determined the expression of 92 the housekeeping genes (ACTB and GAPDH) which were not statistically significantly 93 induced by poly(I:C). Table S1 shows the fold changes (FC) calculated from the microarray 94 data and the real-time PCR-derived expression. Statistical analysis revealed a high level of 95 correspondence (R=0.929, P<0.0001) between the microarray data and the real-time PCR 96 (Figure S1). FCs were logtranformed because of such large variances. 7 97 REFERENCES 98 99 1. Wagener AH, Zwinderman AH, Luiten S, Fokkens WJ, Bel EH, Sterk PJ, van Drunen 100 CM. The impact of allergic rhinitis and asthma on human nasal and bronchial 101 epithelial gene expression. PLoS One, in press. 8 102 Table S1. Validatory PCR of housekeeping genes and significantly different genes. Bronchial epithelial cells Healthy Allergic rhinitis Allergic rhinitis & asthma PCR FC Microarray FC PCR FC Microarray FC PCR FC Microarray FC ACTB 1.16 -1.03 1.52 1.04 1.38 -1.11 GAPDH ATF3 -1.83 -1.00 -1.82 -1.03 -1.49 -1.03 1.43 2.37 -1.78 1.21 -2.10 1.51 CXCL10 DUSP1 EREG 568.10 263.55 214.38 121.34 170.31 88.92 5.78 6.71 4.01 2.35 4.96 4.16 1.37 2.07 -3.59 -1.83 -1.26 1.17 IL13RA2 10.59 14.62 7.32 8.66 7.41 13.44 IL1B IL8 2.83 3.52 1.30 1.75 2.43 3.11 32.48 13.98 44.45 23.40 26.06 16.60 PDE4D -3.63 -1.33 -6.19 -1.15 -3.81 -1.33 TIMP2 1.50 2.67 1.29 2.39 1.52 2.56 103 Nasal epithelial cells Healthy PCR FC 105 Allergic rhinitis & asthma Microarray FC PCR FC Microarray FC PCR FC Microarray FC ACTB -1.43 -1.04 1.04 -1.05 -1.03 -1.06 GAPDH ATF3 CXCL10 DUSP1 EREG -1.92 -1.04 -2.45 -1.06 -1.77 -1.02 9.61 5.02 4.35 3.97 1.69 2.75 465.72 74.79 587.32 175.30 161.27 72.35 11.45 17.60 7.48 15.22 5.98 7.17 3.90 3.13 1.67 3.24 1.03 1.35 IL13RA2 22.16 12.13 17.20 14.39 6.15 5.83 5.25 3.33 3.02 4.04 4.14 3.75 IL1B IL8 104 Allergic rhinitis 178.73 73.41 122.11 64.77 63.85 23.72 PDE4D -1.09 -1.03 -7.87 -1.29 -1.86 -1.04 TIMP2 3.02 4.25 1.77 5.73 1.31 2.97 PCR expression is given as fold change (FC). 9 106 Table S2. dsRNA-induced genes in upper and lower airways Probesets P<0.05 Probesets P<0.05 &FC>4 Genes P<0.05 Genes P<0.05 & FC>4 107 Healthy Nose Healthy Bronchus Rhinitis Nose Rhinitis Bronchus Asthma Nose Asthma Bronchus 17402 13424 15538 7517 6996 8560 1255 550 1439 517 517 427 10163 ↑4636 ↓5527 8342 ↑3638 ↓4704 9353 ↑4204 ↓5149 5190 ↑2294 ↓2896 4919 ↑2293 ↓2626 5810 ↑2621 ↓3189 894 ↑528 ↓366 401 ↑274 ↓127 1037 ↑586 ↓451 391 ↑244 ↓147 383 ↑275 ↓108 319 ↑227 ↓92 FC=fold change; P=p-value adjusted for multiple testing 10 108 Table S3A. Genes assigned to GO-cluster response to virus induced in the upper airways Gene symbol C7orf25 /// PSMA2 CFL1 LILRB1 PIM2 POLR3F ACTA2 BANF1 BECN1 BNIP3L CCL22 CREBZF CYP1A1 DNAJC3 EEF1G /// TUT1 ENO1 FOSL1 HBXIP IFNAR1 IL12A IL28B LYST MAVS POLR3A POLR3H POLR3K TBK1 ABCE1 ACE2 AP1S1 APOBEC3F APOBEC3G BCL2 BCL3 BNIP3 BST2 C19orf2 CCDC130 CCL4 CCL5 CCT5 DDX58 DUOX2 EIF2AK2 GPAM GTF2F1 HERC5 HNRNPUL1 HSPB1 IFI16 IFI35 IFI44 IFIH1 IFITM1 IFITM2 IFITM3 IFNAR2 IFNB1 IFNE Venn diagram A A A A A B B B B B B B B B B B B B B B B B B B B B C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C FC Healthy 1.30 -1.17 1.21 -1.32 -1.22 -1.27 -1.56 -1.25 1.53 1.42 -1.57 -2.60 1.95 -1.77 -1.33 3.39 -1.23 1.53 2.64 4.53 1.99 -2.00 1.53 -2.61 -2.95 1.83 -3.91 15.24 -1.73 2.78 11.38 -1.24 3.08 2.05 45.73 -1.60 1.40 255.13 517.58 -3.44 25.21 3.01 4.09 -4.25 1.31 46.50 -1.35 -1.99 1.70 38.88 64.88 18.03 8.15 2.92 2.27 3.40 3.17 -1.88 FC Rhinitis FC Asthma -1.34 -1.59 -1.25 1.76 1.33 -1.53 -1.73 1.81 -1.85 -1.38 3.90 -1.32 1.39 2.96 7.30 1.73 -1.69 1.65 -2.44 -2.52 1.49 -4.42 12.25 -1.63 2.80 10.82 -1.40 3.17 2.05 79.99 -1.90 1.41 243.83 717.90 -3.23 38.03 3.86 6.12 -4.77 1.55 72.10 -1.37 -2.25 1.59 67.89 164.77 22.18 10.90 2.99 2.25 3.13 2.77 -2.68 -2.48 7.55 -1.40 2.29 5.06 -1.46 2.43 1.66 27.17 -1.43 1.27 39.32 148.81 -2.21 14.32 2.03 3.56 -2.70 1.33 26.64 -1.27 -1.80 1.74 27.76 42.51 10.42 6.25 2.45 1.93 2.38 1.77 -2.45 11 IFNGR1 IFNGR2 IFNK IL23A IL28A IL29 IL6 IRF3 IRF7 IRF9 ISG15 ISG20 ITCH IVNS1ABP MST1R MX1 MX2 MYD88 NLRC5 ODC1 PCBP2 PLSCR1 POLR3B POLR3C POLR3D POLR3E POLR3G PRKRA PVR RELA RPS15A RSAD2 SAMHD1 SIVA1 STAT1 STAT2 STMN1 TICAM1 TLR3 TNF TRIM11 TRIM22 TRIM25 TRIM5 UNC13D UNC93B1 XPO1 XPR1 ZC3HAV1 ZNF175 ABCC9 CLU DMBT1 MICA /// MICB PSMA2 PTPRC IRAK3 CDK6 C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C E E E E E E F G 1.82 2.45 3.23 20.52 13.29 25.01 20.34 1.57 28.26 2.87 23.50 32.65 2.67 -5.44 2.95 30.17 115.45 2.60 38.61 -2.23 -1.49 6.38 -1.44 1.42 1.70 -2.14 -1.87 -1.90 1.93 2.38 -8.15 260.58 16.36 -2.16 13.66 3.50 -5.68 3.14 11.82 8.16 1.73 4.90 6.43 3.81 1.60 2.40 -1.51 -1.76 16.13 1.67 1.48 2.36 4.05 24.06 15.78 20.48 11.13 1.56 40.73 3.41 46.89 41.94 3.02 -6.14 3.39 92.06 287.26 2.98 38.77 -1.78 -1.56 8.28 -1.63 1.30 1.91 -2.32 -2.15 -2.11 1.81 2.45 -9.98 638.34 31.76 -2.48 17.43 4.78 -4.89 3.12 10.00 6.72 1.78 5.16 8.67 3.90 1.76 3.27 -1.63 -2.49 18.83 1.80 1.23 -1.26 1.44 1.21 -1.27 1.24 -1.82 1.68 1.99 3.67 6.43 4.49 7.40 10.02 1.40 16.88 2.94 15.63 14.82 2.43 -2.57 2.04 21.00 72.32 2.37 12.56 -2.04 -1.38 5.22 -1.45 1.27 1.55 -1.67 -1.36 -1.61 1.54 1.98 -4.19 98.63 12.09 -1.57 10.47 3.40 -2.96 2.38 6.68 4.38 1.59 4.61 4.13 2.94 1.53 2.33 -1.41 -1.23 9.78 1.32 1.38 -1.41 12 109 Venn diagram: A=healthy specific; B=overlap healthy–rhinitis; C=overlap all groups; 110 D=overlap healthy–asthma; E=rhinitis specific; F=overlap rhinitis–asthma; G=asthma 111 specific. FC = fold change. 13 112 Table S3B. Genes assigned to GO-cluster response to virus induced in the lower airways Gene symbol BANF1 BECN1 BNIP3L CXCL12 ENO1 IRF3 MAVS MST1R POLR3D POLR3G PSMA2 RNASEL SIVA1 XPR1 BNIP3 IFNAR1 IFNB1 IFNGR1 IL28B LYST POLR3E POLR3K ZNF175 ABCE1 APOBEC3F APOBEC3G BCL3 BST2 C19orf2 CCL4 CCL5 CCT5 CDK6 CREBZF CYP1A1 DDX58 DNAJC3 DUOX2 EEF1G /// TUT1 EIF2AK2 GPAM HERC5 HNRNPUL1 HSPB1 IFI35 IFI44 IFIH1 IFITM1 IFITM2 IFITM3 IFNAR2 IFNGR2 IL23A IL28A IL29 IL6 IRF7 IRF9 Venn diagram A A A A A A A A A A A A A A B B B B B B B B B C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C C FC Healthy -1.36 -1.23 1.30 -1.20 -1.21 1.31 -1.47 1.37 1.40 -1.62 -1.30 -1.35 -1.73 -1.45 1.55 1.23 1.60 1.64 2.40 1.73 -1.62 -2.11 1.54 -2.06 1.90 6.62 2.44 24.79 -1.42 83.19 736.54 -2.10 -1.46 -1.29 -1.91 28.49 1.48 9.56 -1.53 4.63 -2.90 35.87 -1.26 -1.53 48.10 236.92 18.21 14.95 3.33 2.97 2.43 2.25 9.34 4.21 6.80 8.80 26.30 4.44 FC Rhinitis FC Asthma 1.69 1.42 1.47 1.50 2.16 1.68 -1.93 -2.00 1.88 -2.49 1.76 5.28 2.45 48.02 -1.56 20.90 322.77 -1.80 -1.30 -1.36 -2.85 23.14 1.36 4.80 -1.57 5.40 -2.26 25.29 -1.27 -1.74 39.73 70.93 6.27 10.61 3.65 3.53 2.54 1.87 12.37 4.48 3.95 4.92 19.88 3.44 -1.89 2.11 8.11 1.98 13.00 -1.43 35.57 231.87 -1.85 -1.52 -1.38 -2.10 18.03 1.40 7.87 -1.38 3.60 -2.44 27.95 -1.25 -1.61 31.80 104.82 12.76 11.08 3.48 2.73 2.36 2.01 7.90 4.99 4.96 7.60 20.32 3.67 14 113 ISG15 ISG20 ITCH IVNS1ABP MX1 MX2 MYD88 NLRC5 ODC1 PCBP2 PLSCR1 POLR3A PRKRA RELA RPS15A RSAD2 SAMHD1 STAT1 STAT2 TBK1 TICAM1 TLR3 TNF TRIM22 TRIM25 TRIM5 UNC13D UNC93B1 ZC3HAV1 ACE2 ACTA2 AP1S1 BCL2 CCDC130 FOSL1 GTF2F1 IFI16 IRAK3 NLRP3 POLR3H PVR STMN1 TRIM11 XPO1 IFNE CLU C C C C C C C C C C C C C C C C C C C C C C C C C C C C C D D D D D D D D D D D D D D D E G 49.88 10.51 1.93 -2.43 140.44 201.46 2.17 17.88 -2.02 -1.29 7.77 1.49 -1.59 1.91 -4.45 363.02 17.56 16.34 3.40 1.44 2.30 6.70 4.81 6.91 3.96 2.60 1.50 2.52 12.00 6.49 -1.36 -1.46 -1.43 1.26 2.40 1.25 1.50 1.40 1.77 -1.74 1.65 -2.24 1.59 -1.25 64.58 7.46 1.75 -2.16 175.11 163.72 2.32 12.59 -2.87 -1.29 7.54 1.61 -1.41 1.62 -4.65 304.55 3.38 6.17 2.97 1.42 2.15 3.71 3.95 9.76 3.13 2.55 1.43 1.82 15.83 32.63 8.46 1.86 -2.07 67.81 96.81 2.19 11.61 -2.11 -1.31 5.70 1.29 -1.57 1.69 -3.17 167.47 13.59 11.13 2.59 1.30 2.31 5.54 4.35 4.63 3.72 2.51 1.45 2.21 8.50 5.12 -1.38 -1.34 -1.42 1.23 1.89 1.29 1.62 1.56 1.46 -1.45 1.60 -1.93 1.54 -1.31 -2.22 -1.35 Venn diagram: A=healthy specific; B=overlap healthy–rhinitis; C=overlap all groups; 114 D=overlap healthy–asthma; E=rhinitis specific; F=overlap rhinitis–asthma; G=asthma 115 specific. FC = fold change. 15 116 Table S4. Genes induced in the upper airways of healthy controls and allergic rhinitis 117 patients, assigned to GO cluster Mitochondrion. Gene alias Gene name/description AARS2 ABCA12 ABCF2 ACACB ACAD8 ACAD9 ACADM ACADVL ACN9 ACO2 ACOT13 ACP6 ACSF2 ACSL1 ACSS1 ADCK4 ADH5 ADO AIFM1 AK2 AK3 AKAP10 AKT2 ALDH1B1 ALDH1L1 ALDH1L2 ALDH2 ALDH5A1 ALKBH1 ALKBH7 AP2M1 APOA1BP ARG2 ARMC10 ATIC ATP5B ATP5C1 ATP5D ATP5G1 ATP5G2 ATP5G3 ATP5H ATP5I ATP5J ATP5J2 ATP5L ATP5S ATP5SL ATPAF1 BAX BBOX1 alanyl-tRNA synthetase 2, mitochondrial (putative) ATP-binding cassette, sub-family A (ABC1), member 12 ATP-binding cassette, sub-family F (GCN20), member 2 acetyl-CoA carboxylase beta acyl-CoA dehydrogenase family, member 8 acyl-CoA dehydrogenase family, member 9 acyl-CoA dehydrogenase, C-4 to C-12 straight chain acyl-CoA dehydrogenase, very long chain ACN9 homolog (S. cerevisiae) aconitase 2, mitochondrial acyl-CoA thioesterase 13 acid phosphatase 6, lysophosphatidic acyl-CoA synthetase family member 2 acyl-CoA synthetase long-chain family member 1 acyl-CoA synthetase short-chain family member 1 aarF domain containing kinase 4 alcohol dehydrogenase 5 (class III), chi polypeptide 2-aminoethanethiol (cysteamine) dioxygenase apoptosis-inducing factor, mitochondrion-associated, 1 adenylate kinase 2 adenylate kinase 3 A kinase (PRKA) anchor protein 10 v-akt murine thymoma viral oncogene homolog 2 aldehyde dehydrogenase 1 family, member B1 aldehyde dehydrogenase 1 family, member L1 aldehyde dehydrogenase 1 family, member L2 aldehyde dehydrogenase 2 family (mitochondrial) aldehyde dehydrogenase 5 family, member A1 alkB, alkylation repair homolog 1 (E. coli) alkB, alkylation repair homolog 7 (E. coli) adaptor-related protein complex 2, mu 1 subunit apolipoprotein A-I binding protein arginase, type II armadillo repeat containing 10 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide ATP synthase, H+ transporting, mitochondrial F1 complex, gamma polypeptide 1 ATP synthase, H+ transporting, mitochondrial F1 complex, delta subunit ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C1 (subunit 9) ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C2 (subunit 9) ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C3 (subunit 9) ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d ATP synthase, H+ transporting, mitochondrial F0 complex, subunit E ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F6 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F2 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit G ATP synthase, H+ transporting, mitochondrial F0 complex, subunit s (factor B) ATP5S-like ATP synthase mitochondrial F1 complex assembly factor 1 BCL2-associated X protein butyrobetaine (gamma), 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 16 BCL2L1 BCS1L BDH2 BNIP3L BOLA1 BPHL BRP44 C10orf2 C10orf58 C12orf10 C12orf62 C14orf156 C14orf159 C15orf62 C17orf61 C17orf90 C18orf19 C19orf70 C1orf151 C20orf24 C21orf33 C2orf47 C2orf64 C3orf1 C3orf31 C4orf46 /// TOMM7 C5orf54 C6orf57 C7orf55 C7orf55 /// LUC7L2 C9orf46 CA5B CA5BP CAV1 CBARA1 CCBL2 CCDC123 CCDC58 CCT7 CDS2 /// LOC149832 CECR5 CHCHD1 CHCHD2 CHCHD7 CISD2 CISD3 CKMT1A /// CKMT1B CLN3 COL4A3BP COMTD1 COQ10A COQ3 COQ5 COQ6 COQ9 COX10 COX15 COX4I1 COX4NB BCL2-like 1 BCS1-like (yeast) 3-hydroxybutyrate dehydrogenase, type 2 BCL2/adenovirus E1B 19kDa interacting protein 3-like bolA homolog 1 (E. coli) biphenyl hydrolase-like (serine hydrolase) brain protein 44 chromosome 10 open reading frame 2 chromosome 10 open reading frame 58 chromosome 12 open reading frame 10 chromosome 12 open reading frame 62 chromosome 14 open reading frame 156 chromosome 14 open reading frame 159 chromosome 15 open reading frame 62 chromosome 17 open reading frame 61 chromosome 17 open reading frame 90 chromosome 18 open reading frame 19 chromosome 19 open reading frame 70 chromosome 1 open reading frame 151 chromosome 20 open reading frame 24 chromosome 21 open reading frame 33 chromosome 2 open reading frame 47 chromosome 2 open reading frame 64 chromosome 3 open reading frame 1 Chromosome 3 open reading frame 31 chromosome 4 open reading frame 46 /// translocase of outer mitochondrial membrane 7 homolog (yeast) chromosome 5 open reading frame 54 chromosome 6 open reading frame 57 chromosome 7 open reading frame 55 chromosome 7 open reading frame 55 /// LUC7-like 2 (S. cerevisiae) chromosome 9 open reading frame 46 carbonic anhydrase VB, mitochondrial Carbonic anhydrase VB pseudogene caveolin 1, caveolae protein, 22kDa calcium binding atopy-related autoantigen 1 cysteine conjugate-beta lyase 2 coiled-coil domain containing 123 coiled-coil domain containing 58 chaperonin containing TCP1, subunit 7 (eta) CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase) 2 cat eye syndrome chromosome region, candidate 5 coiled-coil-helix-coiled-coil-helix domain containing 1 coiled-coil-helix-coiled-coil-helix domain containing 2 coiled-coil-helix-coiled-coil-helix domain containing 7 CDGSH iron sulfur domain 2 CDGSH iron sulfur domain 3 creatine kinase, mitochondrial 1A /// creatine kinase, mitochondrial 1B ceroid-lipofuscinosis, neuronal 3 collagen, type IV, alpha 3 (Goodpasture antigen) binding protein catechol-O-methyltransferase domain containing 1 coenzyme Q10 homolog A (S. cerevisiae) coenzyme Q3 homolog, methyltransferase (S. cerevisiae) coenzyme Q5 homolog, methyltransferase (S. cerevisiae) coenzyme Q6 homolog, monooxygenase (S. cerevisiae) coenzyme Q9 homolog (S. cerevisiae) COX10 homolog, cytochrome c oxidase assembly protein, heme A: farnesyltransferase (yeast) COX15 homolog, cytochrome c oxidase assembly protein (yeast) cytochrome c oxidase subunit IV isoform 1 COX4 neighbor 17 COX5B COX6B1 COX6C COX7A2L COX7B COX7C CPOX CPS1 CPT2 CRAT CS CYB5B CYC1 CYP1A1 CYP24A1 DAB1 /// OMA1 DARS2 DBI DECR1 DHODH DHRS4 /// DHRS4L2 DLD DLST DNAJA3 DNAJC19 DNLZ DNM3 DRG2 DYNLL1 EARS2 ECH1 EHHADH ELAC2 ENDOG ENO1 ETFA ETFB FAHD1 FAM110B FAM136A FAM175A FAM82B FANCG FARS2 FASTK FDPS FDX1 FECH FEZ1 FIBP FIS1 FUNDC2 FXC1 FXN GABBR1 /// UBD GAD1 GADD45GIP1 GAS8 cytochrome c oxidase subunit Vb cytochrome c oxidase subunit VIb polypeptide 1 (ubiquitous) cytochrome c oxidase subunit VIc cytochrome c oxidase subunit VIIa polypeptide 2 like cytochrome c oxidase subunit VIIb cytochrome c oxidase subunit VIIc coproporphyrinogen oxidase carbamoyl-phosphate synthase 1, mitochondrial carnitine palmitoyltransferase 2 carnitine O-acetyltransferase citrate synthase cytochrome b5 type B (outer mitochondrial membrane) cytochrome c-1 cytochrome P450, family 1, subfamily A, polypeptide 1 cytochrome P450, family 24, subfamily A, polypeptide 1 disabled homolog 1 (Drosophila) /// OMA1 homolog, zinc metallopeptidase (S. cerevisiae) aspartyl-tRNA synthetase 2, mitochondrial diazepam binding inhibitor (GABA receptor modulator, acyl-CoA binding protein) 2,4-dienoyl CoA reductase 1, mitochondrial dihydroorotate dehydrogenase dehydrogenase/reductase (SDR family) member 4 /// dehydrogenase/reductase (SDR family) member 4 like 2 dihydrolipoamide dehydrogenase dihydrolipoamide S-succinyltransferase (E2 component of 2-oxo-glutarate complex) DnaJ (Hsp40) homolog, subfamily A, member 3 DnaJ (Hsp40) homolog, subfamily C, member 19 DNL-type zinc finger dynamin 3 developmentally regulated GTP binding protein 2 dynein, light chain, LC8-type 1 glutamyl-tRNA synthetase 2, mitochondrial (putative) enoyl CoA hydratase 1, peroxisomal enoyl-CoA, hydratase/3-hydroxyacyl CoA dehydrogenase elaC homolog 2 (E. coli) endonuclease G enolase 1, (alpha) electron-transfer-flavoprotein, alpha polypeptide electron-transfer-flavoprotein, beta polypeptide fumarylacetoacetate hydrolase domain containing 1 family with sequence similarity 110, member B family with sequence similarity 136, member A family with sequence similarity 175, member A Family with sequence similarity 82, member B Fanconi anemia, complementation group G Phenylalanyl-tRNA synthetase 2, mitochondrial Fas-activated serine/threonine kinase farnesyl diphosphate synthase (farnesyl pyrophosphate synthetase, dimethylallyltranstransferase, geranyltranstransferase) ferredoxin 1 ferrochelatase fasciculation and elongation protein zeta 1 (zygin I) fibroblast growth factor (acidic) intracellular binding protein fission 1 (mitochondrial outer membrane) homolog (S. cerevisiae) FUN14 domain containing 2 fracture callus 1 homolog (rat) frataxin gamma-aminobutyric acid (GABA) B receptor, 1 /// ubiquitin D glutamate decarboxylase 1 (brain, 67kDa) growth arrest and DNA-damage-inducible, gamma interacting protein 1 growth arrest-specific 8 18 GBAS GCDH GHITM GJA1 GK GK /// GK3P GK3P GLOD4 GLS2 GLT8D1 GLYCTK GM2A GNPAT GPX4 GRAMD4 GRN GTF2H4 GTPBP3 HCCS HDDC2 HEBP1 HEBP2 HEMK1 HIBADH HINT2 HK1 HK2 HLCS HRSP12 HSD17B10 HSD17B8 HSP90AB1 HSPE1 ICT1 IDH1 IDH2 IDH3A IDH3B IMMP2L ISCA1 ISOC2 KARS KIAA0141 KMO KRT5 L2HGDH LARS2 LETM1 LETMD1 LGALS3 LIMK2 LIPT1 LONP1 LRP5 LYRM1 LYRM2 LYRM4 LYRM5 MARS2 glioblastoma amplified sequence glutaryl-CoA dehydrogenase growth hormone inducible transmembrane protein gap junction protein, alpha 1, 43kDa glycerol kinase glycerol kinase /// glycerol kinase 3 pseudogene glycerol kinase 3 pseudogene glyoxalase domain containing 4 glutaminase 2 (liver, mitochondrial) Glycosyltransferase 8 domain containing 1 glycerate kinase GM2 ganglioside activator glyceronephosphate O-acyltransferase glutathione peroxidase 4 (phospholipid hydroperoxidase) GRAM domain containing 4 granulin general transcription factor IIH, polypeptide 4, 52kDa GTP binding protein 3 (mitochondrial) holocytochrome c synthase HD domain containing 2 heme binding protein 1 heme binding protein 2 HemK methyltransferase family member 1 3-hydroxyisobutyrate dehydrogenase histidine triad nucleotide binding protein 2 hexokinase 1 hexokinase 2 holocarboxylase synthetase (biotin-(proprionyl-CoA-carboxylase (ATP-hydrolysing)) ligase) Heat-responsive protein 12 hydroxysteroid (17-beta) dehydrogenase 10 hydroxysteroid (17-beta) dehydrogenase 8 heat shock protein 90kDa alpha (cytosolic), class B member 1 heat shock 10kDa protein 1 (chaperonin 10) immature colon carcinoma transcript 1 Isocitrate dehydrogenase 1 (NADP+), soluble isocitrate dehydrogenase 2 (NADP+), mitochondrial isocitrate dehydrogenase 3 (NAD+) alpha isocitrate dehydrogenase 3 (NAD+) beta IMP2 inner mitochondrial membrane peptidase-like (S. cerevisiae) iron-sulfur cluster assembly 1 homolog (S. cerevisiae) isochorismatase domain containing 2 lysyl-tRNA synthetase KIAA0141 kynurenine 3-monooxygenase (kynurenine 3-hydroxylase) keratin 5 L-2-hydroxyglutarate dehydrogenase leucyl-tRNA synthetase 2, mitochondrial leucine zipper-EF-hand containing transmembrane protein 1 LETM1 domain containing 1 Lectin, galactoside-binding, soluble, 3 LIM domain kinase 2 lipoyltransferase 1 lon peptidase 1, mitochondrial Low density lipoprotein receptor-related protein 5 LYR motif containing 1 LYR motif containing 2 LYR motif containing 4 LYR motif containing 5 methionyl-tRNA synthetase 2, mitochondrial 19 MAT2B MAVS MCAT MCEE MCL1 ME3 MECR MFF MIPEP MLXIP MMACHC MOSC1 MPST MPV17 MPV17L2 MRPL1 MRPL11 MRPL12 MRPL13 MRPL16 MRPL18 MRPL19 MRPL2 MRPL20 MRPL21 MRPL22 MRPL23 MRPL30 MRPL32 MRPL35 MRPL4 MRPL40 MRPL41 MRPL42 MRPL45 MRPL48 MRPL49 MRPL50 MRPL51 /// SPTLC1 MRPL55 MRPL9 MRPS12 MRPS14 MRPS17 /// ZNF713 MRPS18A MRPS2 MRPS21 MRPS23 MRPS25 MRPS27 MRPS28 MRPS31 MRPS33 MRPS34 MRPS7 MRPS9 MSRB2 MSTO1 MTERFD1 Methionine adenosyltransferase II, beta mitochondrial antiviral signaling protein malonyl CoA:ACP acyltransferase (mitochondrial) methylmalonyl CoA epimerase myeloid cell leukemia sequence 1 (BCL2-related) malic enzyme 3, NADP(+)-dependent, mitochondrial mitochondrial trans-2-enoyl-CoA reductase mitochondrial fission factor mitochondrial intermediate peptidase MLX interacting protein methylmalonic aciduria (cobalamin deficiency) cblC type, with homocystinuria MOCO sulphurase C-terminal domain containing 1 mercaptopyruvate sulfurtransferase MpV17 mitochondrial inner membrane protein MPV17 mitochondrial membrane protein-like 2 mitochondrial ribosomal protein L1 mitochondrial ribosomal protein L11 Mitochondrial ribosomal protein L12 mitochondrial ribosomal protein L13 mitochondrial ribosomal protein L16 mitochondrial ribosomal protein L18 mitochondrial ribosomal protein L19 mitochondrial ribosomal protein L2 mitochondrial ribosomal protein L20 mitochondrial ribosomal protein L21 mitochondrial ribosomal protein L22 mitochondrial ribosomal protein L23 mitochondrial ribosomal protein L30 mitochondrial ribosomal protein L32 mitochondrial ribosomal protein L35 mitochondrial ribosomal protein L4 mitochondrial ribosomal protein L40 mitochondrial ribosomal protein L41 mitochondrial ribosomal protein L42 mitochondrial ribosomal protein L45 mitochondrial ribosomal protein L48 mitochondrial ribosomal protein L49 mitochondrial ribosomal protein L50 mitochondrial ribosomal protein L51 /// serine palmitoyltransferase, long chain base subunit 1 mitochondrial ribosomal protein L55 mitochondrial ribosomal protein L9 mitochondrial ribosomal protein S12 mitochondrial ribosomal protein S14 mitochondrial ribosomal protein S17 /// zinc finger protein 713 mitochondrial ribosomal protein S18A mitochondrial ribosomal protein S2 mitochondrial ribosomal protein S21 mitochondrial ribosomal protein S23 mitochondrial ribosomal protein S25 mitochondrial ribosomal protein S27 mitochondrial ribosomal protein S28 mitochondrial ribosomal protein S31 mitochondrial ribosomal protein S33 mitochondrial ribosomal protein S34 mitochondrial ribosomal protein S7 mitochondrial ribosomal protein S9 methionine sulfoxide reductase B2 misato homolog 1 (Drosophila) MTERF domain containing 1 20 MTERFD3 MTG1 MTHFD1L MTIF2 MTOR MTUS1 MUT MUTYH NCRNA00219 NDUFA1 NDUFA10 NDUFA11 NDUFA12 NDUFA13 NDUFA2 NDUFA3 NDUFA4 NDUFA6 NDUFA7 NDUFA8 NDUFA9 NDUFAB1 NDUFAF2 NDUFB3 NDUFB5 NDUFB7 NDUFB8 NDUFB9 NDUFC2 NDUFS5 /// RPL10 NDUFS7 NDUFV1 NDUFV3 NEFH NFS1 NFU1 NIT2 NLRX1 NOP14 NR3C1 NRAS NRD1 NT5DC3 NUDT13 NUDT19 NUDT6 NUDT9 OAT OCIAD1 OGDH OGG1 OMA1 OXA1L OXR1 OXSM P4HA1 PACS2 PARG PARK7 MTERF domain containing 3 mitochondrial GTPase 1 homolog (S. cerevisiae) methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like mitochondrial translational initiation factor 2 mechanistic target of rapamycin (serine/threonine kinase) Microtubule associated tumor suppressor 1 methylmalonyl CoA mutase mutY homolog (E. coli) non-protein coding RNA 219 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 1, 7.5kDa NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 10, 42kDa NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 11, 14.7kDa NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 12 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 13 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 2, 8kDa NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 3, 9kDa NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4, 9kDa NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 6, 14kDa NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 7, 14.5kDa NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 8, 19kDa NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9, 39kDa NADH dehydrogenase (ubiquinone) 1, alpha/beta subcomplex, 1, 8kDa NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, assembly factor 2 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 3, 12kDa NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5, 16kDa NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 7, 18kDa NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 8, 19kDa NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 9, 22kDa NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 2, 14.5kDa NADH dehydrogenase (ubiquinone) Fe-S protein 5, (NADH-coenzyme Q reductase) /// ribosomal protein L10 NADH dehydrogenase (ubiquinone) Fe-S protein 7, 20kDa (NADH-coenzyme Q reductase) NADH dehydrogenase (ubiquinone) flavoprotein 1, 51kDa NADH dehydrogenase (ubiquinone) flavoprotein 3, 10kDa neurofilament, heavy polypeptide NFS1 nitrogen fixation 1 homolog (S. cerevisiae) NFU1 iron-sulfur cluster scaffold homolog (S. cerevisiae) nitrilase family, member 2 NLR family member X1 NOP14 nucleolar protein homolog (yeast) nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) neuroblastoma RAS viral (v-ras) oncogene homolog nardilysin (N-arginine dibasic convertase) 5'-nucleotidase domain containing 3 nudix (nucleoside diphosphate linked moiety X)-type motif 13 nudix (nucleoside diphosphate linked moiety X)-type motif 19 nudix (nucleoside diphosphate linked moiety X)-type motif 6 nudix (nucleoside diphosphate linked moiety X)-type motif 9 ornithine aminotransferase OCIA domain containing 1 oxoglutarate (alpha-ketoglutarate) dehydrogenase (lipoamide) 8-oxoguanine DNA glycosylase OMA1 homolog, zinc metallopeptidase (S. cerevisiae) oxidase (cytochrome c) assembly 1-like oxidation resistance 1 3-oxoacyl-ACP synthase, mitochondrial prolyl 4-hydroxylase, alpha polypeptide I phosphofurin acidic cluster sorting protein 2 poly (ADP-ribose) glycohydrolase Parkinson disease (autosomal recessive, early onset) 7 21 PARL PARS2 PCCB PCK2 PDK1 PDSS2 PECI PEMT PERP PET112L PGAM5 PHYH PHYHIPL PICK1 PIGY PINK1 PITRM1 PMPCA PNKD POLDIP2 POLG POLG2 POLRMT PON2 PPIF PPOX PPP1CA PPP2CA PPP3CB PRDX1 PRDX2 PRDX5 PRKACA PTGR2 PTRF PTRH1 PTRH2 PTS PUS1 PYCR1 QTRT1 QTRTD1 RAF1 RAI14 RARS2 RBFA RDBP RDH13 RG9MTD1 RNF5 RPL9 RPP21 /// TRIM39 /// TRIM39R RPS6KB1 RPUSD4 SAMM50 SARS SCCPDH SDHA presenilin associated, rhomboid-like prolyl-tRNA synthetase 2, mitochondrial (putative) propionyl CoA carboxylase, beta polypeptide phosphoenolpyruvate carboxykinase 2 (mitochondrial) pyruvate dehydrogenase kinase, isozyme 1 prenyl (decaprenyl) diphosphate synthase, subunit 2 peroxisomal D3,D2-enoyl-CoA isomerase phosphatidylethanolamine N-methyltransferase PERP, TP53 apoptosis effector PET112-like (yeast) phosphoglycerate mutase family member 5 phytanoyl-CoA 2-hydroxylase phytanoyl-CoA 2-hydroxylase interacting protein-like protein interacting with PRKCA 1 phosphatidylinositol glycan anchor biosynthesis, class Y PTEN induced putative kinase 1 pitrilysin metallopeptidase 1 peptidase (mitochondrial processing) alpha paroxysmal nonkinesigenic dyskinesia polymerase (DNA-directed), delta interacting protein 2 polymerase (DNA directed), gamma polymerase (DNA directed), gamma 2, accessory subunit polymerase (RNA) mitochondrial (DNA directed) paraoxonase 2 peptidylprolyl isomerase F protoporphyrinogen oxidase protein phosphatase 1, catalytic subunit, alpha isozyme protein phosphatase 2, catalytic subunit, alpha isozyme protein phosphatase 3, catalytic subunit, beta isozyme peroxiredoxin 1 peroxiredoxin 2 peroxiredoxin 5 protein kinase, cAMP-dependent, catalytic, alpha prostaglandin reductase 2 polymerase I and transcript release factor Peptidyl-tRNA hydrolase 1 homolog (S. cerevisiae) peptidyl-tRNA hydrolase 2 6-pyruvoyltetrahydropterin synthase pseudouridylate synthase 1 pyrroline-5-carboxylate reductase 1 queuine tRNA-ribosyltransferase 1 queuine tRNA-ribosyltransferase domain containing 1 v-raf-1 murine leukemia viral oncogene homolog 1 retinoic acid induced 14 arginyl-tRNA synthetase 2, mitochondrial ribosome binding factor A RD RNA binding protein Retinol dehydrogenase 13 (all-trans/9-cis) RNA (guanine-9-) methyltransferase domain containing 1 ring finger protein 5 ribosomal protein L9 ribonuclease P/MRP 21kDa subunit /// tripartite motif-containing 39 /// TRIM39-like protein ribosomal protein S6 kinase, 70kDa, polypeptide 1 RNA pseudouridylate synthase domain containing 4 sorting and assembly machinery component 50 homolog (S. cerevisiae) Seryl-tRNA synthetase saccharopine dehydrogenase (putative) succinate dehydrogenase complex, subunit A, flavoprotein (Fp) 22 SDHA /// SDHAP1 /// SDHAP2 SDHAF1 SDHC SDHD SFXN1 SFXN2 SHC1 SIRT5 SLC1A3 SLC25A12 SLC25A14 SLC25A25 SLC25A26 SLC25A29 SLC25A3 SLC25A32 SLC25A36 SLC25A38 SLC25A39 SLC25A44 SLC25A6 SLC27A3 SLIT3 SLMO2 SNN SOD1 STAR STOML2 SUCLG1 SURF1 TATDN3 TBC1D15 TBRG4 THG1L TIMM13 TIMM17A TIMM22 TIMM23 /// TIMM23B TIMM8A TMEM126A TMEM14B /// TMEM14C TMEM14C TMEM160 TMEM223 TMTC1 TOMM22 TOMM34 TOMM70A TP53 TRAP1 TRIT1 TRNT1 TXNDC12 TXNRD1 UQCC UQCR10 UQCR11 succinate dehydrogenase complex, subunit A, flavoprotein (Fp) /// succinate dehydrogenase complex, subunit A, flavoprotein pseudogene 1 /// succinate dehydrogenase complex, subunit A, flavoprotein pseudogene 2 succinate dehydrogenase complex assembly factor 1 succinate dehydrogenase complex, subunit C, integral membrane protein, 15kDa succinate dehydrogenase complex, subunit D, integral membrane protein sideroflexin 1 sideroflexin 2 SHC (Src homology 2 domain containing) transforming protein 1 sirtuin (silent mating type information regulation 2 homolog) 5 (S. cerevisiae) solute carrier family 1 (glial high affinity glutamate transporter), member 3 solute carrier family 25 (mitochondrial carrier, Aralar), member 12 solute carrier family 25 (mitochondrial carrier, brain), member 14 solute carrier family 25, member 25 solute carrier family 25, member 26 solute carrier family 25, member 29 solute carrier family 25, member 3 solute carrier family 25, member 32 Solute carrier family 25, member 36 solute carrier family 25, member 38 solute carrier family 25, member 39 solute carrier family 25, member 44 solute carrier family 25, member 6 solute carrier family 27 (fatty acid transporter), member 3 slit homolog 3 (Drosophila) slowmo homolog 2 (Drosophila) stannin superoxide dismutase 1, soluble steroidogenic acute regulatory protein stomatin (EPB72)-like 2 succinate-CoA ligase, alpha subunit surfeit 1 TatD DNase domain containing 3 TBC1 domain family, member 15 transforming growth factor beta regulator 4 tRNA-histidine guanylyltransferase 1-like (S. cerevisiae) translocase of inner mitochondrial membrane 13 homolog (yeast) translocase of inner mitochondrial membrane 17 homolog A (yeast) Translocase of inner mitochondrial membrane 22 homolog (yeast) translocase of inner mitochondrial membrane 23 homolog (yeast) /// translocase of inner mitochondrial membrane 23 homolog B (yeast) translocase of inner mitochondrial membrane 8 homolog A (yeast) transmembrane protein 126A transmembrane protein 14B /// transmembrane protein 14C transmembrane protein 14C transmembrane protein 160 transmembrane protein 223 transmembrane and tetratricopeptide repeat containing 1 translocase of outer mitochondrial membrane 22 homolog (yeast) translocase of outer mitochondrial membrane 34 translocase of outer mitochondrial membrane 70 homolog A (S. cerevisiae) tumor protein p53 TNF receptor-associated protein 1 tRNA isopentenyltransferase 1 tRNA nucleotidyl transferase, CCA-adding, 1 thioredoxin domain containing 12 (endoplasmic reticulum) thioredoxin reductase 1 ubiquinol-cytochrome c reductase complex chaperone ubiquinol-cytochrome c reductase, complex III subunit X ubiquinol-cytochrome c reductase, complex III subunit XI 23 UQCRB UQCRH UQCRQ USP30 VARS2 VHL WARS2 WASF1 YME1L1 YWHAE YWHAZ ubiquinol-cytochrome c reductase binding protein ubiquinol-cytochrome c reductase hinge protein ubiquinol-cytochrome c reductase, complex III subunit VII, 9.5kDa ubiquitin specific peptidase 30 valyl-tRNA synthetase 2, mitochondrial (putative) von Hippel-Lindau tumor suppressor tryptophanyl tRNA synthetase 2, mitochondrial WAS protein family, member 1 YME1-like 1 (S. cerevisiae) Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide 24 118 119 Table S5. Genes induced in the lower airways of healthy controls, assigned to GO cluster Mitochondrion. Gene alias Gene name/description ABCB6 ACADVL ACP6 ADH5 ADO AIFM1 AK1 AKT2 ALDH18A1 ALKBH7 ARAF ARSB ASS1 ATP5C1 ATP5G3 ATP5H ATP5I ATP5J2 ATP5SL ATP6V1A AURKAIP1 BCL2L10 BCL2L2 BLOC1S1 BNIP3L C10orf58 C12orf10 C12orf62 C14orf159 C15orf62 C17orf61 C18orf19 C1orf31 C22orf32 C2orf56 C9orf46 CA5B CCDC142 /// MRPL53 CCDC58 CDS2 /// LOC149832 CHAF1B CHCHD1 CHCHD2 CHCHD4 CIDEA CISD3 CLPX CLTC COL4A3BP COMTD1 COQ5 ATP-binding cassette, sub-family B (MDR/TAP), member 6 acyl-CoA dehydrogenase, very long chain acid phosphatase 6, lysophosphatidic alcohol dehydrogenase 5 (class III), chi polypeptide 2-aminoethanethiol (cysteamine) dioxygenase apoptosis-inducing factor, mitochondrion-associated, 1 adenylate kinase 1 v-akt murine thymoma viral oncogene homolog 2 aldehyde dehydrogenase 18 family, member A1 AlkB, alkylation repair homolog 7 (E. coli) v-raf murine sarcoma 3611 viral oncogene homolog arylsulfatase B argininosuccinate synthase 1 ATP synthase, H+ transporting, mitochondrial F1 complex, gamma polypeptide 1 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C3 (subunit 9) ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d ATP synthase, H+ transporting, mitochondrial F0 complex, subunit E ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F2 ATP5S-like ATPase, H+ transporting, lysosomal 70kDa, V1 subunit A aurora kinase A interacting protein 1 BCL2-like 10 (apoptosis facilitator) BCL2-like 2 biogenesis of lysosomal organelles complex-1, subunit 1 BCL2/adenovirus E1B 19kDa interacting protein 3-like chromosome 10 open reading frame 58 chromosome 12 open reading frame 10 chromosome 12 open reading frame 62 chromosome 14 open reading frame 159 chromosome 15 open reading frame 62 chromosome 17 open reading frame 61 chromosome 18 open reading frame 19 chromosome 1 open reading frame 31 chromosome 22 open reading frame 32 chromosome 2 open reading frame 56 chromosome 9 open reading frame 46 carbonic anhydrase VB, mitochondrial coiled-coil domain containing 142 /// mitochondrial ribosomal protein L53 coiled-coil domain containing 58 CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase) 2 chromatin assembly factor 1, subunit B (p60) coiled-coil-helix-coiled-coil-helix domain containing 1 coiled-coil-helix-coiled-coil-helix domain containing 2 coiled-coil-helix-coiled-coil-helix domain containing 4 cell death-inducing DFFA-like effector a CDGSH iron sulfur domain 3 ClpX caseinolytic peptidase X homolog (E. coli) Clathrin, heavy chain (Hc) collagen, type IV, alpha 3 (Goodpasture antigen) binding protein catechol-O-methyltransferase domain containing 1 coenzyme Q5 homolog, methyltransferase (S. cerevisiae) 25 COQ9 COX10 COX15 COX16 COX4NB COX5B COX6B1 COX7C CRAT CROT CXorf23 CYB5R1 CYB5R3 DDX28 DHODH DHX29 DHX30 DLST DNAJC15 DNAJC4 DPYSL2 DRG2 DYNLL1 EHHADH ELAC2 ENDOG ENO1 FAHD1 FASTK FOXRED1 FTSJ2 FUNDC2 FXC1 FXN GADD45GIP1 GATC GATM GHITM GK /// GK3P GK3P GLUL GLYCTK GNPAT GOT2 GRN GRPEL1 GRPEL2 GSTZ1 GTF2H4 GTPBP5 GTPBP8 HEBP2 HEMK1 HK2 HSCB HTRA2 ICT1 IDH1 IDH3B coenzyme Q9 homolog (S. cerevisiae) COX10 homolog, cytochrome c oxidase assembly protein, heme A: farnesyltransferase (yeast) COX15 homolog, cytochrome c oxidase assembly protein (yeast) COX16 cytochrome c oxidase assembly homolog (S. cerevisiae) COX4 neighbor Cytochrome c oxidase subunit Vb cytochrome c oxidase subunit VIb polypeptide 1 (ubiquitous) cytochrome c oxidase subunit VIIc carnitine O-acetyltransferase carnitine O-octanoyltransferase chromosome X open reading frame 23 cytochrome b5 reductase 1 cytochrome b5 reductase 3 DEAD (Asp-Glu-Ala-Asp) box polypeptide 28 dihydroorotate dehydrogenase DEAH (Asp-Glu-Ala-His) box polypeptide 29 DEAH (Asp-Glu-Ala-His) box polypeptide 30 dihydrolipoamide S-succinyltransferase (E2 component of 2-oxo-glutarate complex) DnaJ (Hsp40) homolog, subfamily C, member 15 DnaJ (Hsp40) homolog, subfamily C, member 4 dihydropyrimidinase-like 2 developmentally regulated GTP binding protein 2 dynein, light chain, LC8-type 1 enoyl-CoA, hydratase/3-hydroxyacyl CoA dehydrogenase elaC homolog 2 (E. coli) endonuclease G enolase 1, (alpha) fumarylacetoacetate hydrolase domain containing 1 Fas-activated serine/threonine kinase FAD-dependent oxidoreductase domain containing 1 FtsJ homolog 2 (E. coli) FUN14 domain containing 2 fracture callus 1 homolog (rat) frataxin Growth arrest and DNA-damage-inducible, gamma interacting protein 1 Glutamyl-tRNA(Gln) amidotransferase, subunit C homolog (bacterial) Glycine amidinotransferase (L-arginine:glycine amidinotransferase) growth hormone inducible transmembrane protein glycerol kinase /// glycerol kinase 3 pseudogene glycerol kinase 3 pseudogene glutamate-ammonia ligase glycerate kinase glyceronephosphate O-acyltransferase glutamic-oxaloacetic transaminase 2, mitochondrial (aspartate aminotransferase 2) granulin GrpE-like 1, mitochondrial (E. coli) GrpE-like 2, mitochondrial (E. coli) glutathione transferase zeta 1 general transcription factor IIH, polypeptide 4, 52kDa GTP binding protein 5 (putative) GTP-binding protein 8 (putative) heme binding protein 2 HemK methyltransferase family member 1 hexokinase 2 HscB iron-sulfur cluster co-chaperone homolog (E. coli) HtrA serine peptidase 2 immature colon carcinoma transcript 1 Isocitrate dehydrogenase 1 (NADP+), soluble isocitrate dehydrogenase 3 (NAD+) beta 26 ISCA1 ISCA2 IVD JMJD7 KIAA0141 KIF1B KMO LARS2 LETM1 LETMD1 LGALS3 LIMK2 LOC100510009 /// WBSCR16 LYRM2 MAVS MCAT MCL1 MECR MMACHC MRPL11 MRPL13 MRPL16 MRPL17 MRPL18 MRPL20 MRPL22 MRPL32 MRPL33 MRPL36 MRPL4 MRPL41 MRPL43 MRPL45 MRPL50 MRPL55 MRPL9 MRPS12 MRPS14 MRPS15 MRPS17 /// ZNF713 MRPS2 MRPS24 MRPS26 MRPS28 MRPS9 MSTO1 /// MSTO2P MTFMT MTG1 MTOR MUT NAGS NDUFA1 NDUFA11 NDUFA12 NDUFA13 NDUFA3 NDUFA4 NDUFA8 NDUFAB1 iron-sulfur cluster assembly 1 homolog (S. cerevisiae) iron-sulfur cluster assembly 2 homolog (S. cerevisiae) isovaleryl-CoA dehydrogenase jumonji domain containing 7 KIAA0141 kinesin family member 1B kynurenine 3-monooxygenase (kynurenine 3-hydroxylase) leucyl-tRNA synthetase 2, mitochondrial leucine zipper-EF-hand containing transmembrane protein 1 LETM1 domain containing 1 lectin, galactoside-binding, soluble, 3 LIM domain kinase 2 Williams-Beuren syndrome chromosome region 16 LYR motif containing 2 mitochondrial antiviral signaling protein malonyl CoA:ACP acyltransferase (mitochondrial) myeloid cell leukemia sequence 1 (BCL2-related) mitochondrial trans-2-enoyl-CoA reductase methylmalonic aciduria (cobalamin deficiency) cblC type, with homocystinuria mitochondrial ribosomal protein L11 mitochondrial ribosomal protein L13 mitochondrial ribosomal protein L16 mitochondrial ribosomal protein L17 mitochondrial ribosomal protein L18 mitochondrial ribosomal protein L20 mitochondrial ribosomal protein L22 mitochondrial ribosomal protein L32 mitochondrial ribosomal protein L33 mitochondrial ribosomal protein L36 mitochondrial ribosomal protein L4 mitochondrial ribosomal protein L41 mitochondrial ribosomal protein L43 mitochondrial ribosomal protein L45 mitochondrial ribosomal protein L50 Mitochondrial ribosomal protein L55 mitochondrial ribosomal protein L9 Mitochondrial ribosomal protein S12 mitochondrial ribosomal protein S14 mitochondrial ribosomal protein S15 mitochondrial ribosomal protein S17 /// zinc finger protein 713 mitochondrial ribosomal protein S2 mitochondrial ribosomal protein S24 mitochondrial ribosomal protein S26 mitochondrial ribosomal protein S28 mitochondrial ribosomal protein S9 misato homolog 1 (Drosophila) /// misato homolog 2 pseudogene mitochondrial methionyl-tRNA formyltransferase mitochondrial GTPase 1 homolog (S. cerevisiae) Mechanistic target of rapamycin (serine/threonine kinase) methylmalonyl CoA mutase N-acetylglutamate synthase NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 1, 7.5kDa NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 11, 14.7kDa NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 12 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 13 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 3, 9kDa NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4, 9kDa NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 8, 19kDa NADH dehydrogenase (ubiquinone) 1, alpha/beta subcomplex, 1, 8kDa 27 NDUFB1 NDUFB3 NDUFB5 NDUFB7 NDUFB9 NDUFS5 /// RPL10 NDUFS6 NFS1 NLRX1 NR3C1 NRAS NRD1 NT5C OAT OCIAD1 OXSM P4HA1 PAM16 PANK2 PARG PARL PARS2 PCCA PDK1 PDSS2 PECR PET112L PIN4 PMPCA POLDIP2 PPP1CA PPP1CC PPP3CA PROSC PTRH1 PTRH2 PUS1 RAB11A RAB3D RAI14 RARS2 RBFA RDBP RDH13 RNASEL RNF5 RPP21 /// TRIM39 /// TRIM39R SACS SARDH SARS2 SEC61A1 SFXN5 SH3BP5 SIVA1 SLC1A3 SLC25A13 SLC25A14 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 1, 7kDa NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 3, 12kDa NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5, 16kDa NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 7, 18kDa NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 9, 22kDa NADH dehydrogenase (ubiquinone) Fe-S protein 5, 15kDa (NADH-coenzyme Q reductase) /// ribosomal protein L10 NADH dehydrogenase (ubiquinone) Fe-S protein 6, 13kDa (NADH-coenzyme Q reductase) NFS1 nitrogen fixation 1 homolog (S. cerevisiae) NLR family member X1 nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) neuroblastoma RAS viral (v-ras) oncogene homolog nardilysin (N-arginine dibasic convertase) 5', 3'-nucleotidase, cytosolic ornithine aminotransferase OCIA domain containing 1 3-oxoacyl-ACP synthase, mitochondrial prolyl 4-hydroxylase, alpha polypeptide I presequence translocase-associated motor 16 homolog Pantothenate kinase 2 poly (ADP-ribose) glycohydrolase presenilin associated, rhomboid-like prolyl-tRNA synthetase 2, mitochondrial (putative) Propionyl Coenzyme A carboxylase, alpha polypeptide pyruvate dehydrogenase kinase, isozyme 1 prenyl (decaprenyl) diphosphate synthase, subunit 2 peroxisomal trans-2-enoyl-CoA reductase PET112-like (yeast) protein (peptidylprolyl cis/trans isomerase) NIMA-interacting, 4 (parvulin) peptidase (mitochondrial processing) alpha polymerase (DNA-directed), delta interacting protein 2 protein phosphatase 1, catalytic subunit, alpha isozyme protein phosphatase 1, catalytic subunit, gamma isozyme protein phosphatase 3, catalytic subunit, alpha isozyme Proline synthetase co-transcribed homolog (bacterial) peptidyl-tRNA hydrolase 1 homolog (S. cerevisiae) peptidyl-tRNA hydrolase 2 pseudouridylate synthase 1 RAB11A, member RAS oncogene family RAB3D, member RAS oncogene family retinoic acid induced 14 arginyl-tRNA synthetase 2, mitochondrial ribosome binding factor A RD RNA binding protein Retinol dehydrogenase 13 (all-trans/9-cis) ribonuclease L (2',5'-oligoisoadenylate synthetase-dependent) ring finger protein 5 ribonuclease P/MRP 21kDa subunit /// tripartite motif-containing 39 /// TRIM39-like protein spastic ataxia of Charlevoix-Saguenay (sacsin) Sarcosine dehydrogenase seryl-tRNA synthetase 2, mitochondrial Sec61 alpha 1 subunit (S. cerevisiae) sideroflexin 5 SH3-domain binding protein 5 (BTK-associated) SIVA1, apoptosis-inducing factor solute carrier family 1 (glial high affinity glutamate transporter), member 3 Solute carrier family 25, member 13 (citrin) solute carrier family 25 (mitochondrial carrier, brain), member 14 28 SLC25A25 SLC25A26 SLC25A29 SLC25A30 SLC25A32 SLC25A38 SLC25A44 SLC25A45 SLC27A1 SLC27A3 SLMO2 SND1 SOD1 SRGAP2 SSSCA1 STAR SURF1 SYNJ2BP TAOK3 TATDN3 TBC1D15 TBRG4 TDRKH TIMM17A TIMM23 TMEM126A TMEM14B /// TMEM14C TMEM14C TMEM186 TMEM223 TOMM40 TXNIP UQCR11 UQCRC2 UQCRFS1 WARS2 ZFHX3 solute carrier family 25, member 25 solute carrier family 25, member 26 Solute carrier family 25, member 29 solute carrier family 25, member 30 solute carrier family 25, member 32 solute carrier family 25, member 38 solute carrier family 25, member 44 solute carrier family 25, member 45 solute carrier family 27 (fatty acid transporter), member 1 solute carrier family 27 (fatty acid transporter), member 3 slowmo homolog 2 (Drosophila) staphylococcal nuclease and tudor domain containing 1 superoxide dismutase 1, soluble SLIT-ROBO Rho GTPase activating protein 2 Sjogren syndrome/scleroderma autoantigen 1 steroidogenic acute regulatory protein surfeit 1 synaptojanin 2 binding protein TAO kinase 3 TatD DNase domain containing 3 TBC1 domain family, member 15 transforming growth factor beta regulator 4 tudor and KH domain containing translocase of inner mitochondrial membrane 17 homolog A (yeast) translocase of inner mitochondrial membrane 23 homolog (yeast) transmembrane protein 126A transmembrane protein 14B /// transmembrane protein 14C transmembrane protein 14C transmembrane protein 186 transmembrane protein 223 translocase of outer mitochondrial membrane 40 homolog (yeast) thioredoxin interacting protein ubiquinol-cytochrome c reductase, complex III subunit XI ubiquinol-cytochrome c reductase core protein II ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 tryptophanyl tRNA synthetase 2, mitochondrial Zinc finger homeobox 3 29 120 121 Table S6. Genes induced in the lower airways of healthy controls and allergic rhinitis patients, assigned to GO cluster Mitochondrion. Gene alias Gene name/description AASS ADCK2 AK3 AK4 ATP5J ATP5L BBOX1 BCAT1 BNIP3 C10orf2 C14orf156 C19orf70 C2orf47 C3orf1 C3orf78 C4orf14 C6orf203 C7orf55 /// LUC7L2 CCBL2 CCT7 CKB CKMT1A /// CKMT1B COX11 COX3 COX7A2L CPS1 CS CYCS DARS2 DHRS1 DLAT EFHD1 FIS1 GCAT GCDH GLRX2 HERC2 KARS KRT4 LONP1 LYRM4 MARS2 MCCC2 METT11D1 METTL12 MMAA MPST MRPL12 MRPL2 MRPL21 MRPL35 aminoadipate-semialdehyde synthase aarF domain containing kinase 2 adenylate kinase 3 adenylate kinase 4 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F6 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit G butyrobetaine (gamma), 2-oxoglutarate dioxygenase (gamma-butyrobetaine hydroxylase) 1 branched chain amino-acid transaminase 1, cytosolic BCL2/adenovirus E1B 19kDa interacting protein 3 chromosome 10 open reading frame 2 chromosome 14 open reading frame 156 chromosome 19 open reading frame 70 chromosome 2 open reading frame 47 chromosome 3 open reading frame 1 small integral membrane protein 4 chromosome 4 open reading frame 14 chromosome 6 open reading frame 203 chromosome 7 open reading frame 55 /// LUC7-like 2 (S. cerevisiae) cysteine conjugate-beta lyase 2 chaperonin containing TCP1, subunit 7 (eta) creatine kinase, brain creatine kinase, mitochondrial 1A /// creatine kinase, mitochondrial 1B COX11 cytochrome c oxidase assembly homolog (yeast) mitochondrially encoded cytochrome c oxidase III cytochrome c oxidase subunit VIIa polypeptide 2 like carbamoyl-phosphate synthase 1, mitochondrial citrate synthase cytochrome c, somatic aspartyl-tRNA synthetase 2, mitochondrial dehydrogenase/reductase (SDR family) member 1 dihydrolipoamide S-acetyltransferase EF-hand domain family, member D1 fission 1 (mitochondrial outer membrane) homolog (S. cerevisiae) glycine C-acetyltransferase glutaryl-CoA dehydrogenase glutaredoxin 2 hect domain and RLD 2 lysyl-tRNA synthetase keratin 4 lon peptidase 1, mitochondrial LYR motif containing 4 methionyl-tRNA synthetase 2, mitochondrial Methylcrotonoyl-Coenzyme A carboxylase 2 (beta) methyltransferase like 17 methyltransferase like 12 methylmalonic aciduria (cobalamin deficiency) cblA type mercaptopyruvate sulfurtransferase mitochondrial ribosomal protein L12 mitochondrial ribosomal protein L2 mitochondrial ribosomal protein L21 mitochondrial ribosomal protein L35 30 MRPL42 MRPS16 MRPS23 MRPS25 MRPS33 MRPS34 MRPS35 MRPS7 MSTO1 MTERFD1 MTHFD2 MTPAP NAPG NDUFA9 NDUFB8 NDUFC1 NDUFS7 NDUFV3 NIPSNAP1 NIT2 NOP14 NR1D1 /// THRA NUDT19 NUDT9 PITRM1 POLG2 PPOX PRDX1 QTRTD1 RARS REXO2 SFXN4 SLC22A4 SLC25A15 SLC25A19 SMCR7L SUPV3L1 TFB1M TFB2M TIMM44 TK2 TMEM160 TMLHE TOMM70A TXNDC12 UQCRB UQCRQ VARS YME1L1 YWHAE mitochondrial ribosomal protein L42 mitochondrial ribosomal protein S16 mitochondrial ribosomal protein S23 mitochondrial ribosomal protein S25 mitochondrial ribosomal protein S33 mitochondrial ribosomal protein S34 mitochondrial ribosomal protein S35 mitochondrial ribosomal protein S7 misato homolog 1 (Drosophila) MTERF domain containing 1 methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase mitochondrial poly(A) polymerase N-ethylmaleimide-sensitive factor attachment protein, gamma NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9, 39kDa NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 8, 19kDa NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 1, 6kDa NADH dehydrogenase (ubiquinone) Fe-S protein 7, 20kDa (NADH-coenzyme Q reductase) NADH dehydrogenase (ubiquinone) flavoprotein 3, 10kDa nipsnap homolog 1 (C. elegans) nitrilase family, member 2 NOP14 nucleolar protein homolog (yeast) nuclear receptor subfamily 1, group D, member 1 /// thyroid hormone receptor, alpha (erythroblastic leukemia viral (v-erb-a) oncogene homolog, avian) nudix (nucleoside diphosphate linked moiety X)-type motif 19 nudix (nucleoside diphosphate linked moiety X)-type motif 9 pitrilysin metallopeptidase 1 polymerase (DNA directed), gamma 2, accessory subunit protoporphyrinogen oxidase peroxiredoxin 1 queuine tRNA-ribosyltransferase domain containing 1 arginyl-tRNA synthetase REX2, RNA exonuclease 2 homolog (S. cerevisiae) Sideroflexin 4 solute carrier family 22 (organic cation/ergothioneine transporter), member 4 solute carrier family 25 (mitochondrial carrier; ornithine transporter), member 15 solute carrier family 25 (mitochondrial thiamine pyrophosphate carrier), member 19 Smith-Magenis syndrome chromosome region, candidate 7-like suppressor of var1, 3-like 1 (S. cerevisiae) transcription factor B1, mitochondrial transcription factor B2, mitochondrial translocase of inner mitochondrial membrane 44 homolog (yeast) thymidine kinase 2, mitochondrial transmembrane protein 160 trimethyllysine hydroxylase, epsilon translocase of outer mitochondrial membrane 70 homolog A (S. cerevisiae) thioredoxin domain containing 12 (endoplasmic reticulum) ubiquinol-cytochrome c reductase binding protein ubiquinol-cytochrome c reductase, complex III subunit VII, 9.5kDa valyl-tRNA synthetase YME1-like 1 (S. cerevisiae) tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide 31 122 Figure legends 123 Figure S1. Correlation plot of real-time PCR data and microarray results. FCs were 124 logtranformed.