Supplementary Data

Chemical genomics identifies the unfolded response as a target for selective cancer cell killing during glucose deprivation

Sakae Saito1, Aki Furuno1, Junko Sakurai1, Asami Sakamoto1, Hae-Ryong Park2, Kazuo

Shin-ya3, Takashi Tsuruo1, Akihiro Tomida1

1 Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo, Japan

2 Department of Food Science and Biotechnology, Kyungnam University, Masan,

Korea.

3 Biological Information Research Center, National Institute of Advanced Industrial

Science and Technology, Tokyo, Japan

- 1 - Supplemental Methods

Reporter assay. The reporter assay was performed as described previously (1).

HT1080 cells were cultured overnight in a 12-well plate (3 u 105 cells/well), and transfection was performed. The cells were incubated for 8 hours with a transfection mixture containing 500 ng of firefly luciferase–containing reporter plasmids

(pGRP78pro160-Luc(1) or FLAG-tagged XBP1-Luc) and 1 ng of renilla luciferase–containing plasmid phRL-CMV (Promega, WI, USA) as an internal control.

The medium was then replaced with fresh growth medium, and the cells were incubated for another 4 hours. Subsequently, the cells were reseeded in a 96-well plate (5 u 103 cells/well), cultured overnight, and treated for 18 hours with various concentrations of versipelostatin (VST), biguanides or pyrvinium pamoate with or without 10 mM

2-deoxy-D-glucose (2DG) or 5 Pg/mL of Tunicamycin (TM). Relative activity of firefly luciferase to renilla luciferase was determined using the Dual-Glo Luciferase

Assay System (Promega).

Immunoblot analysis. Cells were lysed in 1u sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, and 10% glycerol), and protein concentrations of the lysates were measured with a Bio-Rad protein assay kit (Bio-Rad, CA, USA). Equal amounts of were resolved on a 4%–20%

SDS-polyacrylamide gradient gel and transferred by electroblotting onto a nitrocellulose membrane. Immunoblots were probed with the following antibodies: mouse monoclonal anti-KDEL (for detection of GRP78 and GRP94; StressGen, BC, Canada), rabbit polyclonal anti-eIF2D, anti-phospho-eIF2D (Ser51), rabbit monoclonal anti-AMPKD, anti-phospho-AMPKD (Thr172) (Cell Signaling Technology, Inc., MA,

- 2 - USA), rabbit polyclonal anti-CREB-2 (ATF4) (Santa Cruz Biotechnology, Inc., CA,

USA), anti-FLAG M2 (to detect FLAG-tagged ATF6 proteins; Sigma) and anti-E-actin

(internal control; Sigma). Rabbit polyclonal anti-EIF2AK3 (PERK) antibody was raised against synthetic peptide corresponding to the cytoplasmic domain

CKDESTDWPLSSPSP of the human PERK protein. The specific signals were detected with horseradish peroxidase–conjugated second antibody (Amersham

Pharmacia Biotech, Tokyo, Japan) and a chemiluminescence detection system

(PerkinElmer, Inc., MA, USA).

Microarray expression profiles. Total RNA from cultured cells was isolated using the RNeasy RNA purification kit (Qiagen, CA, USA). The quality of total RNA was analyzed using the RNA 6000 Nano LabChip kit on a 2100 Bioanalyzer (Agilent

Technologies, CA, USA). cRNA targets for hybridization to GeneChip were prepared by reverse transcription from 5 Pg of total RNA. Targets were then labeled with biotin before fragmentation according to standard Affymetrix protocols. Hybridization to

GeneChip U133 Plus 2.0 arrays (Affymetrix) was carried out using

Fluidics Station 450 and GeneChip Scanner 3000 (Affymetrix).

Quantitative real-time PCR. Total RNA from HeLa cell lines was isolated by using the RNeasy RNA purification kit (Qiagen) with DNase I treatment to digest genomic

DNA contamination. As a reference, QPCR Reference Human Total RNA (Stratagene,

CA, USA) was used. First-strand cDNAs were synthesized by reverse transcription from 5 Pg of total RNA using the SuperScrip II First-Strand Synthesis System

(Invitrogen) primed with oligo-(dT)12-18 and random hexamers according to the manufacturer’s instructions. The quantitative real-time PCR was then performed with

FAM-labeled D-LUX primer sets (Invitrogen) in 16 selected from the UPR-CS

- 3 - signature genes. The specific primers were designed with the D-LUX Designer software (Invitogen), and E-actin was used as a control in all experiments. All of the primer sequences are described to Supplemental Table S4. The PCR reaction mixtures were set up with Platinum Quantitative PCR SuperMix-UDG (Invitrogen) according to the manufacturer’s instructions. Each 20 Pl reaction contains 100 nM of each -specific primer and the template cDNA (2 nM). The two-step PCR cycling condition consisted of incubation at 50 qC for 15 min, then at 95 qC for 2 min, followed by 45 cycles each at 95 qC for 15 sec and 62 qC for 30 sec. Reactions were conducted in the 96-well spectrofluorometric thermal cycler ABI PRISM 7700 (Applied

Biosystems, CA, USA). Fluorescence was monitored during every PCR cycle at the annealing step.

- 4 - Supplemental Table S1. Summary of the experimental conditions for 42 microarray samples. profiles were determined with Affymetrix Human Genome U133 plus 2.0 microarrays in 4 cancer cell lines. The cells were treated with VST, biguanides or pyruvinium pamoate in adequate dosage under normal or ER stress conditions for 15 or 18 hours. The exposure time periods were sufficient to activate the UPR in those cells but not to allow apparent overt cell death, even in the presence of UPR modulators. A total of 42 samples were classified into 4 groups: cells under normal growth conditions (control, black), under ER stress conditions (stress, red), under ER stress conditions with a UPR modulator (stress+ modulator, blue) and under normal conditions with a UPR modulator (modulator, green). GF, glucose-free; 2-DG, 2-deoxyglucose; TM, tunicamycin; VST, versipelostatin; PP, pyrvinium pamoate.

HeLa HT-29 HT1080 MKN74 Control 18h(1), 15h(2) 18h(1) (2) 18h(1) (2) (3) (6) 18h VST 1PM,18h 10PM,18h 3PM,18h 1PM,18h Buformin ņņ 300PM,18h ņ Metformin ņņ 10mM,18h ņ Phenformin ņņ 100PM,18h ņ PP ņņ 0.1PM, 18h ņ GF 18h(1), 15h(2) 18h(1) (2) 18h 18h GF/VST 1PM,18h 10PM,18h 3PM,18h 1PM,18h GF/Buformin 50PM,15h ņņņ GF/Metformin 1.5mM,15h ņņņ GF/Phenformin 15PM,15h ņņņ 2DG 5mM,18h ņ 10mM,18h(1) (4) ņ 2DG/VST 5mM/1PM, 18h ņ 10mM/3PM,18h ņ 2DG/Buformin ņņ 10mM/300PM, 18h ņ 2DG/Metformin ņņ 10mM/10mM, 18h ņ 2DG/Phenformin ņņ 10mM/100PM, 18h ņ 2DG/PP ņņ 10mM/0.1PM, 18h ņ TM 1Pg/mL,18h ņ 5Pg/mL,18h ņ TM/VST ņņ 5Pg/mL /3PM,18h ņ

- 5 - Supplemental Table S2. List of CEL files. To identify the Glucose Deprivation signature genes and the VST/biguanides signature genes, RMA and SAM analysis was performed with .respectively ,(غ) and 10 samples (ع) data sets from 16 samples

Sample Name .CEL file Paired/Baseline SAM غع HeLa_Cont UPR_HeLa_Cont.CEL ņ ع HeLa_Cont-2 UPR_HeLa_Cont-2.CEL ņ ع HeLa_GF UPR_HeLa_GF.CEL HeLa_Cont غ HeLa_VST UPR_HeLa_VST.CEL HeLa_Cont HeLa_GF+VST UPR_HeLa_GF+VST.CEL HeLa_Cont ع HeLa_2DG UPR_HeLa_2DG.CEL HeLa_Cont-2 HeLa_TM UPR_HeLa_TM.CEL HeLa_Cont-2 HeLa_GF-2 UPR_HeLa_GF-2.CEL HeLa_Cont-2 HeLa_2DG+VST UPR_HeLa_2DG+VST.CEL HeLa_Cont-2 HeLa_GF+Bu UPR_HeLa_GF+Bu.CEL HeLa_Cont-2 HeLa_GF+Met UPR_HeLa_GF+Met.CEL HeLa_Cont-2 HeLa_GF+Phen UPR_HeLa_GF+Phen.CEL HeLa_Cont-2 غع HT-29_Cont UPR_HT-29_Cont.CEL ņ ع HT-29_Cont-2 UPR_HT-29_Cont-2.CEL ņ ع HT-29_GF UPR_HT-29_GF.CEL HT-29_Cont غ HT-29_VST UPR_HT-29_VST.CEL HT-29_Cont HT-29_GF+VST UPR_HT-29_GF+VST.CEL HT-29_Cont غع HT-29_GF-2 UPR_HT-29_GF-2.CEL HT-29_Cont-2 ع HT1080_Cont UPR_HT1080_Cont.CEL ņ ع HT1080_Cont-2 UPR_HT1080_Cont-2.CEL ņ غ HT1080_Cont-3 UPR_HT1080_Cont-3.CEL ņ ع HT1080_Cont-6 UPR_HT1080_Cont-6.CEL ņ ع HT1080_GF UPR_HT1080_GF.CEL HT1080_Cont غ HT1080_VST UPR_HT1080_VST.CEL HT1080_Cont HT1080_GF+VST UPR_HT1080_GF+VST.CEL HT1080_Cont ع HT1080_2DG UPR_HT1080_2DG.CEL HT1080_Cont-2 HT1080_PP UPR_HT1080_PP.CEL HT1080_Cont-2 HT1080_2DG+PP UPR_HT1080_2DG+PP.CEL HT1080_Cont-2 HT1080_Bu UPR_HT1080_Bu.CEL HT1080_Cont-3 HT1080_Met UPR_HT1080_Met.CEL HT1080_Cont-3 غ HT1080_Phen UPR_HT1080_Phen.CEL HT1080_Cont-3 HT1080_2DG+Bu UPR_HT1080_2DG+Bu.CEL HT1080_Cont-3 HT1080_2DG+Met UPR_HT1080_2DG+Met.CEL HT1080_Cont-3 HT1080_2DG+Phen UPR_HT1080_2DG+Phen.CEL HT1080_Cont-3 ع HT1080_2DG-4 UPR_HT1080_2DG-4.CEL HT1080_Cont-6 HT1080_TM UPR_HT1080_TM.CEL HT1080_Cont-6 HT1080_2DG+VST UPR_HT1080_2DG+VST.CEL HT1080_Cont-6 HT1080_TM+VST UPR_HT1080_TM+VST.CEL HT1080_Cont-6 غع MKN74_Cont UPR_MKN74_Cont.CEL ņ غ MKN74_VST UPR_MKN74_VST.CEL MKN74_Cont ع MKN74_GF UPR_MKN74_GF.CEL MKN74_Cont MKN74_GF+VST UPR_MKN74_GF+VST.CEL MKN74_Cont

- 6 - Supplemental Table S3. List of the Glucose Deprivation signature genes. The annotation data was obtained from NetAffyx (Affymetrix). Data shown are sorted by Score (d) from SAM analysis.

A. Up-regulated 148 probe sets (97 genes) Score Fold q-value Probe Set ID Gene Symbol Gene Title (d) Change (%) 1558080_s_at LOC144871 hypothetical protein LOC144871 15.667 3.65 0 230265_at SEL1L Sel-1 suppressor of lin-12-like (C. elegans) 14.395 4.00 0 208499_s_at DNAJC3 DnaJ (Hsp40) homolog, subfamily C, member 3 13.925 3.73 0 202061_s_at SEL1L sel-1 suppressor of lin-12-like (C. elegans) 13.159 4.30 0 235341_at DNAJC3 DnaJ (Hsp40) homolog, subfamily C, member 3 13.033 3.33 0 203252_at CDK2AP2 CDK2-associated protein 2 12.927 2.26 0 heat shock 70kDa protein 5 (glucose-regulated 211936_at HSPA5 12.834 2.71 0 protein, 78kDa) 225284_at LOC144871 hypothetical protein LOC144871 12.546 2.56 0 200825_s_at HYOU1 hypoxia up-regulated 1 12.123 3.85 0 homocysteine-inducible, endoplasmic reticulum 217168_s_at HERPUD1 12.055 5.81 0 stress-inducible, ubiquitin-like domain member 1 202062_s_at SEL1L sel-1 suppressor of lin-12-like (C. elegans) 12.051 4.00 0 202842_s_at DNAJB9 DnaJ (Hsp40) homolog, subfamily B, member 9 11.945 6.78 0 210592_s_at SAT1 spermidine/spermine N1-acetyltransferase 1 11.763 3.11 0 203455_s_at SAT1 spermidine/spermine N1-acetyltransferase 1 11.701 3.17 0 solute carrier family 33 (acetyl-CoA transporter), 203165_s_at SLC33A1 11.517 3.11 0 member 1 213988_s_at SAT1 spermidine/spermine N1-acetyltransferase 1 11.503 2.72 0 phosphatidic acid phosphatase type 2 domain 223568_s_at PPAPDC1B 11.203 2.45 0 containing 1B phosphatidic acid phosphatase type 2 domain 226150_at PPAPDC1B 10.619 3.04 0 containing 1B 202655_at ARMET arginine-rich, mutated in early stage tumors 10.491 3.25 0 solute carrier family 33 (acetyl-CoA transporter), 203164_at SLC33A1 10.347 2.34 0 member 1 220606_s_at C17orf48 17 open reading frame 48 9.937 2.02 0 chondroitin sulfate 218871_x_at CSGALNACT2 9.818 3.01 0 N-acetylgalactosaminyltransferase 2 202064_s_at SEL1L sel-1 suppressor of lin-12-like (C. elegans) 9.744 4.07 0 ubiquitin-conjugating enzyme E2, J1 (UBC6 217826_s_at UBE2J1 9.741 2.24 0 homolog, yeast) 202843_at DNAJB9 DnaJ (Hsp40) homolog, subfamily B, member 9 9.645 12.22 0 224841_x_at GAS5 growth arrest-specific 5 9.630 2.28 0 epidermal growth factor receptor (erythroblastic 201984_s_at EGFR 9.565 2.58 0 leukemia viral (v-erb-b) oncogene homolog, avian) solute carrier family 33 (acetyl-CoA transporter), 1554148_a_at SLC33A1 9.539 2.77 0 member 1 227033_at PDIA3 protein disulfide isomerase family A, member 3 9.306 2.57 0 solute carrier family 39 (zinc transporter), member 212110_at SLC39A14 9.299 2.06 0 14 1554462_a_at DNAJB9 DnaJ (Hsp40) homolog, subfamily B, member 9 9.215 7.41 0 ubiquitin-conjugating enzyme E2, J1 (UBC6 217823_s_at UBE2J1 9.163 2.01 0 homolog, yeast) 218681_s_at SDF2L1 stromal cell-derived factor 2-like 1 9.137 3.63 0 224741_x_at GAS5 growth arrest-specific 5 9.072 2.20 0 ubiquitin-conjugating enzyme E2, J1 (UBC6 222435_s_at UBE2J1 8.891 2.16 0 homolog, yeast)

- 7 - 223054_at DNAJB11 DnaJ (Hsp40) homolog, subfamily B, member 11 8.817 2.75 0 221782_at DNAJC10 DnaJ (Hsp40) homolog, subfamily C, member 10 8.758 2.48 0 225090_at SYVN1 synovial apoptosis inhibitor 1, synoviolin 8.495 2.31 0 230659_at --- Transcribed 8.487 2.25 0 heat shock 70kDa protein 5 (glucose-regulated 230031_at HSPA5 8.483 5.47 0 protein, 78kDa) 221865_at C9orf91 chromosome 9 open reading frame 91 8.193 2.21 0 223789_s_at GTPBP2 GTP binding protein 2 8.123 3.72 0 200935_at CALR calreticulin 8.090 2.52 0 219600_s_at TMEM50B transmembrane protein 50B 8.080 2.49 0 208613_s_at FLNB filamin B, beta (actin binding protein 278) 8.074 2.14 0 236285_at --- Transcribed locus 8.041 6.27 0 218145_at TRIB3 tribbles homolog 3 (Drosophila) 7.994 4.59 0 200670_at XBP1 X-box binding protein 1 7.906 2.76 0 229453_at PDIA3 Protein disulfide isomerase family A, member 3 7.835 2.47 0 226835_s_at C20orf199 chromosome 20 open reading frame 199 7.784 2.11 0 216985_s_at STX3 syntaxin 3 7.779 2.17 0 211048_s_at PDIA4 protein disulfide isomerase family A, member 4 7.727 3.31 0.106 218358_at CRELD2 cysteine-rich with EGF-like domains 2 7.676 4.07 0.106 phosphatidic acid phosphatase type 2 domain 226384_at PPAPDC1B 7.655 3.16 0.106 containing 1B 209383_at DDIT3 DNA-damage-inducible transcript 3 7.596 14.44 0.106 220012_at ERO1LB ERO1-like beta (S. cerevisiae) 7.541 3.45 0.106 phosphatidic acid phosphatase type 2 domain 223569_at PPAPDC1B 7.532 2.26 0.106 containing 1B Cas-Br-M (murine) ecotropic retroviral transforming 209682_at CBLB 7.486 2.95 0.106 sequence b 229838_at NUCB2 nucleobindin 2 7.428 3.13 0.106 223299_at SEC11C SEC11 homolog C (S. cerevisiae) 7.426 2.18 0.106 219910_at FICD FIC domain containing 7.417 2.73 0.106 223209_s_at SELS selenoprotein S 7.359 2.19 0.106 214316_x_at --- Transcribed locus 7.330 2.02 0.106 chondroitin sulfate 222235_s_at CSGALNACT2 7.317 3.18 0.106 N-acetylgalactosaminyltransferase 2 203675_at NUCB2 nucleobindin 2 7.261 6.20 0.106 200599_s_at HSP90B1 heat shock protein 90kDa beta (Grp94), member 1 7.228 2.47 0.106 204203_at CEBPG CCAAT/enhancer binding protein (C/EBP), gamma 7.093 3.41 0.106 208612_at PDIA3 protein disulfide isomerase family A, member 3 7.082 2.19 0.106 SEC24 related gene family, member A (S. 244841_at SEC24A 7.079 2.09 0.106 cerevisiae) procollagen-proline, 2-oxoglutarate 4-dioxygenase 200656_s_at P4HB 7.078 2.17 0.106 (proline 4-hydroxylase), beta polypeptide procollagen-proline, 2-oxoglutarate 4-dioxygenase 1564494_s_at P4HB 7.053 2.23 0.106 (proline 4-hydroxylase), beta polypeptide Cas-Br-M (murine) ecotropic retroviral transforming 227900_at CBLB 7.034 2.10 0.106 sequence b MAGT1 /// 221553_at magnesium transporter 1 /// hypothetical LOC728866 7.032 2.04 0.106 LOC728866 transmembrane emp24 protein transport domain 214658_at TMED7 7.028 2.09 0.106 containing 7 225956_at C5orf41 chromosome 5 open reading frame 41 7.023 5.61 0.106 1555788_a_at TRIB3 tribbles homolog 3 (Drosophila) 6.968 5.90 0.106 chondroitin sulfate 239077_at CSGALNACT2 6.950 2.21 0.106 N-acetylgalactosaminyltransferase 2 224915_x_at C20orf199 chromosome 20 open reading frame 199 6.882 2.05 0.164 SEC24 related gene family, member A (S. 212902_at SEC24A 6.863 2.60 0.164 cerevisiae) 216640_s_at PDIA6 protein disulfide isomerase family A, member 6 6.820 2.04 0.164

- 8 - 227027_at --- Clone 25028 mRNA sequence 6.757 3.33 0.164 222728_s_at JOSD3 Josephin domain containing 3 6.715 2.06 0.164 1553055_a_at SLFN5 schlafen family member 5 6.619 4.36 0.164 202720_at TES testis derived transcript (3 LIM domains) 6.606 2.76 0.164 222632_s_at LZTFL1 leucine zipper transcription factor-like 1 6.539 2.61 0.164 221050_s_at GTPBP2 GTP binding protein 2 6.520 3.04 0.164 221577_x_at GDF15 growth differentiation factor 15 6.515 5.26 0.164 RAPGEF6 /// Rap guanine nucleotide exchange factor (GEF) 6 /// 228250_at 6.502 2.06 0.188 FNIP1 folliculin interacting protein 1 216268_s_at JAG1 jagged 1 (Alagille syndrome) 6.484 2.95 0.188 202129_s_at RIOK3 RIO kinase 3 (yeast) 6.435 2.46 0.188 203935_at ACVR1 activin A receptor, type I 6.369 2.10 0.188 200598_s_at HSP90B1 heat shock protein 90kDa beta (Grp94), member 1 6.367 3.41 0.188 209743_s_at ITCH itchy E3 ubiquitin protein ligase homolog (mouse) 6.358 2.02 0.188 202722_s_at GFPT1 glutamine-fructose-6-phosphate transaminase 1 6.345 2.73 0.188 221781_s_at DNAJC10 DnaJ (Hsp40) homolog, subfamily C, member 10 6.337 2.05 0.188 208639_x_at PDIA6 protein disulfide isomerase family A, member 6 6.248 2.03 0.188 solute carrier family 1 (glutamate/neutral amino acid 212810_s_at SLC1A4 6.179 5.32 0.188 transporter), member 4 216449_x_at HSP90B1 heat shock protein 90kDa beta (Grp94), member 1 6.177 3.18 0.188 201000_at AARS alanyl-tRNA synthetase 6.163 2.00 0.188 ADAM metallopeptidase domain 17 (tumor necrosis 213532_at ADAM17 6.147 2.12 0.249 factor, alpha, converting enzyme) 202131_s_at RIOK3 RIO kinase 3 (yeast) 6.122 2.64 0.249 thioredoxin domain containing 4 (endoplasmic 208959_s_at TXNDC4 6.113 2.03 0.249 reticulum) eukaryotic translation initiation factor 2-alpha kinase 218696_at EIF2AK3 6.101 3.17 0.249 3 207668_x_at PDIA6 protein disulfide isomerase family A, member 6 6.098 2.09 0.249 203279_at EDEM1 ER degradation enhancer, mannosidase alpha-like 1 6.083 2.19 0.249 213836_s_at WIPI1 WD repeat domain, phosphoinositide interacting 1 6.070 9.19 0.249 209270_at LAMB3 laminin, beta 3 6.028 2.54 0.249 208763_s_at TSC22D3 TSC22 domain family, member 3 6.007 2.49 0.249 208658_at PDIA4 protein disulfide isomerase family A, member 4 5.978 2.92 0.249 1552639_at KLHDC7B kelch domain containing 7B 5.954 2.62 0.249 227755_at --- CDNA clone IMAGE:4077090 5.931 5.98 0.249 229618_at SNX16 sorting nexin 16 5.864 2.08 0.249 FAM18B2 /// family with sequence similarity 18, member B2 /// 228482_at 5.862 2.13 0.249 CDRT4 CMT1A duplicated region transcript 4 inhibitor of Bruton agammaglobulinemia tyrosine 210970_s_at IBTK 5.833 2.34 0.249 kinase 228846_at MXD1 MAX dimerization protein 1 5.823 4.96 0.249 202721_s_at GFPT1 glutamine-fructose-6-phosphate transaminase 1 5.807 2.71 0.307 226886_at --- Clone 25028 mRNA sequence 5.778 2.49 0.307 222907_x_at TMEM50B transmembrane protein 50B 5.737 2.31 0.307 225957_at C5orf41 chromosome 5 open reading frame 41 5.734 3.10 0.307 LysM, putative peptidoglycan-binding, domain 226321_at LYSMD3 5.725 2.24 0.307 containing 3 epidermal growth factor receptor (erythroblastic 201983_s_at EGFR 5.724 2.13 0.307 leukemia viral (v-erb-b) oncogene homolog, avian) 240277_at --- Transcribed locus 5.675 2.00 0.307 209435_s_at ARHGEF2 rho/rac guanine nucleotide exchange factor (GEF) 2 5.674 2.27 0.307 202719_s_at TES testis derived transcript (3 LIM domains) 5.647 3.22 0.307 203827_at WIPI1 WD repeat domain, phosphoinositide interacting 1 5.623 9.00 0.307 227443_at C9orf150 chromosome 9 open reading frame 150 5.543 5.63 0.307 200802_at SARS seryl-tRNA synthetase 5.533 2.26 0.307 223059_s_at FAM107B family with sequence similarity 107, member B 5.517 2.82 0.307

- 9 - 224999_at --- Placenta mRNA, clone PL45, partial sequence 5.499 2.05 0.307 209744_x_at ITCH itchy E3 ubiquitin protein ligase homolog (mouse) 5.460 2.04 0.307 209238_at STX3 syntaxin 3 5.456 2.14 0.307 221156_x_at CCPG1 cell cycle progression 1 5.434 3.21 0.452 209099_x_at JAG1 jagged 1 (Alagille syndrome) 5.425 2.70 0.452 200945_s_at SEC31A SEC31 homolog A (S. cerevisiae) 5.418 2.05 0.452 solute carrier family 1 (glutamate/neutral amino acid 209610_s_at SLC1A4 5.352 4.84 0.452 transporter), member 4 221985_at KLHL24 kelch-like 24 (Drosophila) 5.319 3.39 0.452 stress 70 protein chaperone, microsome-associated, 202558_s_at STCH 5.314 3.68 0.452 60kDa 210999_s_at GRB10 growth factor receptor-bound protein 10 5.304 2.67 0.452 218050_at UFM1 ubiquitin-fold modifier 1 5.299 2.11 0.452 231944_at ERO1LB ERO1-like beta (S. cerevisiae) 5.287 2.90 0.452 SGK3 /// serum/glucocorticoid regulated kinase family, 220038_at 5.279 2.53 0.452 C8orf44 member 3 /// chromosome 8 open reading frame 44 221511_x_at CCPG1 cell cycle progression 1 5.251 2.44 0.452 222690_s_at TMEM39A transmembrane protein 39A 5.238 2.28 0.452 223070_at SELK selenoprotein K 5.220 2.26 0.452 223058_at FAM107B family with sequence similarity 107, member B 5.195 2.74 0.452 solute carrier family 1 (glutamate/neutral amino acid 212811_x_at SLC1A4 5.150 3.71 0.452 transporter), member 4 218972_at TTC17 tetratricopeptide repeat domain 17 5.147 3.24 0.452 218615_s_at TMEM39A transmembrane protein 39A 5.137 2.44 0.452

B. Down-regulated 98 probe sets (81 genes) Score Fold q-value Probe Set ID Gene Symbol Gene Title (d) Change (%) proteasome (prosome, macropain) activator subunit 3 200988_s_at PSME3 -12.032 0.48 0 (PA28 gamma; Ki) 217645_at C14orf112 chromosome 14 open reading frame 112 -11.639 0.45 0 228773_at --- Transcribed locus -11.048 0.46 0 201129_at SFRS7 splicing factor, arginine/serine-rich 7, 35kDa -10.500 0.47 0 227578_at LOC100128191 hypothetical protein LOC100128191 -10.282 0.42 0 1552470_a_at ABHD11 abhydrolase domain containing 11 -10.149 0.43 0 cyclin-dependent kinase inhibitor 3 (CDK2-associated 1555758_a_at CDKN3 -10.135 0.44 0 dual specificity phosphatase) 212020_s_at MKI67 antigen identified by monoclonal antibody Ki-67 -10.045 0.38 0 224509_s_at RTN4IP1 reticulon 4 interacting protein 1 -10.024 0.46 0 cyclin-dependent kinase inhibitor 3 (CDK2-associated 209714_s_at CDKN3 -9.949 0.42 0 dual specificity phosphatase) proteasome (prosome, macropain) subunit, alpha type, 201316_at PSMA2 -9.551 0.49 0 2 203145_at SPAG5 sperm associated antigen 5 -9.400 0.49 0 DLEU2 /// deleted in lymphocytic leukemia, 2 /// deleted in 215629_s_at -8.825 0.36 0 DLEU2L lymphocytic leukemia 2-like 205436_s_at H2AFX H2A histone family, member X -8.474 0.44 0 212023_s_at MKI67 antigen identified by monoclonal antibody Ki-67 -8.129 0.37 0 Acidic (leucine-rich) nuclear phosphoprotein 32 201038_s_at ANP32A -8.038 0.50 0 family, member A cyclin-dependent kinase inhibitor 2C (p18, inhibits 204159_at CDKN2C -7.943 0.38 0 CDK4) 239252_at --- Transcribed locus -7.916 0.44 0

- 10 - 226775_at ENY2 enhancer of yellow 2 homolog (Drosophila) -7.826 0.39 0 SPC24, NDC80 kinetochore complex component, 235572_at SPC24 -7.659 0.49 0 homolog (S. cerevisiae) 209709_s_at HMMR hyaluronan-mediated motility receptor (RHAMM) -7.612 0.45 0 203744_at HMGB3 high-mobility group box 3 -7.282 0.49 0.141 204603_at EXO1 exonuclease 1 -7.281 0.47 0.141 inhibitor of DNA binding 1, dominant negative 208937_s_at ID1 -7.197 0.34 0.141 helix-loop-helix protein 229129_at --- Transcribed locus -7.105 0.45 0.141 228987_at --- Transcribed locus -6.951 0.33 0.141 204444_at KIF11 kinesin family member 11 -6.931 0.42 0.141 221436_s_at CDCA3 cell division cycle associated 3 -6.900 0.39 0.141 207165_at HMMR hyaluronan-mediated motility receptor (RHAMM) -6.884 0.48 0.141 216870_x_at DLEU2 deleted in lymphocytic leukemia, 2 -6.870 0.40 0.141 229305_at MLF1IP MLF1 interacting protein -6.847 0.49 0.141 1555679_a_at RTN4IP1 reticulon 4 interacting protein 1 -6.842 0.46 0.141 processing of precursor 1, ribonuclease P/MRP 213449_at POP1 -6.805 0.46 0.141 subunit (S. cerevisiae) 208955_at DUT deoxyuridine triphosphatase -6.795 0.50 0.141 214331_at TSFM Ts translation elongation factor, mitochondrial -6.780 0.49 0.141 221591_s_at FAM64A family with sequence similarity 64, member A -6.727 0.46 0.141 209377_s_at HMGN3 high mobility group nucleosomal binding domain 3 -6.727 0.45 0.141 202094_at BIRC5 baculoviral IAP repeat-containing 5 (survivin) -6.689 0.41 0.141 225108_at ------6.678 0.40 0.141 polymerase (RNA) III (DNA directed) polypeptide K, 218866_s_at POLR3K -6.666 0.46 0.141 12.3 kDa 235545_at DEPDC1 DEP domain containing 1 -6.632 0.48 0.141 223307_at CDCA3 cell division cycle associated 3 -6.624 0.42 0.141 SPC25, NDC80 kinetochore complex component, 209891_at SPC25 -6.623 0.39 0.141 homolog (S. cerevisiae) ASF1 anti-silencing function 1 homolog B (S. 218115_at ASF1B -6.623 0.45 0.141 cerevisiae) 201969_at NASP nuclear autoantigenic sperm protein (histone-binding) -6.619 0.37 0.141 225687_at FAM83D family with sequence similarity 83, member D -6.589 0.42 0.141 201896_s_at PSRC1 proline/serine-rich coiled-coil 1 -6.587 0.36 0.141 218755_at KIF20A kinesin family member 20A -6.473 0.38 0.164 224865_at MLSTD2 male sterility domain containing 2 -6.462 0.44 0.164 209773_s_at RRM2 ribonucleotide reductase M2 polypeptide -6.447 0.47 0.164 221520_s_at CDCA8 cell division cycle associated 8 -6.428 0.49 0.164 217856_at RBM8A RNA binding motif protein 8A -6.383 0.47 0.164 202240_at PLK1 polo-like kinase 1 (Drosophila) -6.346 0.46 0.164 204827_s_at CCNF cyclin F -6.313 0.37 0.164 221727_at SUB1 SUB1 homolog (S. cerevisiae) -6.126 0.49 0.164 228899_at LOC100132884 hypothetical protein LOC100132884 -6.050 0.45 0.188 asp (abnormal spindle) homolog, microcephaly 219918_s_at ASPM -6.014 0.49 0.188 associated (Drosophila) 203418_at CCNA2 cyclin A2 -6.014 0.39 0.188 213852_at RBM8A RNA binding motif protein 8A -5.998 0.49 0.188 215030_at GRSF1 G-rich RNA sequence binding factor 1 -5.901 0.44 0.188 208079_s_at AURKA aurora kinase A -5.770 0.48 0.188 201890_at RRM2 ribonucleotide reductase M2 polypeptide -5.768 0.47 0.188

- 11 - 203626_s_at SKP2 S-phase kinase-associated protein 2 (p45) -5.670 0.33 0.249 1557487_at --- CDNA clone IMAGE:4797099 -5.630 0.44 0.249 200685_at SFRS11 splicing factor, arginine/serine-rich 11 -5.612 0.50 0.249 218726_at HJURP Holliday junction recognition protein -5.609 0.49 0.249 238431_at --- Transcribed locus -5.597 0.49 0.249 242890_at --- CDNA FLJ34225 fis, clone FCBBF3023372 -5.534 0.36 0.249 204067_at SUOX sulfite oxidase -5.497 0.50 0.249 228045_at --- CDNA FLJ30370 fis, clone BRACE2007832 -5.469 0.49 0.249 220085_at HELLS helicase, lymphoid-specific -5.442 0.42 0.249 215731_s_at MPHOSPH9 M-phase phosphoprotein 9 -5.359 0.46 0.249 asp (abnormal spindle) homolog, microcephaly 232238_at ASPM -5.357 0.49 0.249 associated (Drosophila) 221965_at MPHOSPH9 M-phase phosphoprotein 9 -5.325 0.49 0.307 203276_at LMNB1 lamin B1 -5.312 0.42 0.307 227896_at BCCIP BRCA2 and CDKN1A interacting protein -5.234 0.47 0.307 229126_at TMEM19 transmembrane protein 19 -5.213 0.49 0.307 224428_s_at CDCA7 cell division cycle associated 7 -5.205 0.43 0.307 203625_x_at SKP2 S-phase kinase-associated protein 2 (p45) -5.204 0.35 0.307 211814_s_at CCNE2 cyclin E2 -5.140 0.42 0.307 asp (abnormal spindle) homolog, microcephaly 239002_at ASPM -5.114 0.48 0.307 associated (Drosophila) Ubiquitin B (UBB) mRNA, 3' UTR and genetic 242300_at --- -5.100 0.50 0.307 suppressor element splicing factor, arginine/serine-rich 1 (splicing factor 227164_at SFRS1 -5.060 0.49 0.307 2, alternate splicing factor) 211042_x_at MCAM melanoma cell adhesion molecule -5.043 0.36 0.307 minichromosome maintenance complex component 220651_s_at MCM10 -5.024 0.41 0.307 10 minichromosome maintenance complex component 222962_s_at MCM10 -5.017 0.38 0.307 10 excision repair cross-complementing rodent repair 219650_at ERCC6L -4.984 0.44 0.452 deficiency, complementation group 6-like 215942_s_at GTSE1 G-2 and S-phase expressed 1 -4.984 0.44 0.452 238529_at LOC730631 Hypothetical LOC730631 -4.968 0.46 0.452 227349_at HELLS helicase, lymphoid-specific -4.917 0.45 0.452 213226_at CCNA2 cyclin A2 -4.909 0.47 0.452 minichromosome maintenance complex component 223570_at MCM10 -4.870 0.46 0.452 10 205909_at POLE2 polymerase (DNA directed), epsilon 2 (p59 subunit) -4.821 0.40 0.452 210567_s_at SKP2 S-phase kinase-associated protein 2 (p45) -4.803 0.29 0.452 219990_at E2F8 E2F transcription factor 8 -4.752 0.31 0.452 221521_s_at GINS2 GINS complex subunit 2 (Psf2 homolog) -4.743 0.44 0.452 227350_at HELLS helicase, lymphoid-specific -4.729 0.45 0.452 212170_at RBM12 RNA binding motif protein 12 -4.715 0.48 0.452

- 12 - Supplemental Table S4. Primer sequence of the quantitative real-time PCR. FAM-labeled D-LUX primer sets (Invitrogen) were designed with the D-LUX Designer software (Invitogen). A commercially available house-keeping gene primer set (E-actin) was used as the control in all experiments.

Representative Amplicon Gene Symbol Primer seqence 1 (Fowerd) Primer sequence 2 (Reverse) Gene Title Probe Set ID Public ID size

ARMET NM_006010 TGTCACATTCTCACCAGCCACT CGGTTCCCGATATAGTAGCACAAC[FAM]G 99 arginine-rich, mutated in early stage tumors 202655_at

214316_x_at//212952_at CALR NM_004343 CGGGACCCAGAAATTGACAACCC[FAM]G GCCCAGCACGCCAAAGTTAT 78 Calreticulin //200935_at

CDK2AP2 NM_005851 CGGATGAGATGGGCAAAGAGATC[FAM]G TACCTCTCTTCAGGCGCTCCA 75 CDK2-associated protein 2 203252_at

DNAJB11 NM_016306 TGAAGAACGAACGCTGGAAGTAG CGGCTAAAGGGGTACTCCATGC[FAM]G 69 DnaJ (Hsp40) homolog, subfamily B, member 11 223054_at

DNAJB9 AL080081 CCATGAAGTACCACCCTGACAA CGGATTGAATTTTGCTTCAGCATC[FAM]G 59 DnaJ (Hsp40) homolog, subfamily B, member 9 202842_s_at

DNAJC3 NM_006260 CGTCCGCCACTGCTTTTCTAAGGA[FAM]G GGCATTCACATTGTCAGGTTCC 94 DnaJ (Hsp40) homolog, subfamily C, member 3 208499_s_at

HSPA5 AF216292 CGAGCTTGTTCTTGTTGGTGGCT[FAM]G GGTTTATGCCACGGGATGGT 98 heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) 211936_at

HYOU1 NM_006389 GTGGAGTTCACGAGGGAGGTG CGGGCCATCCGAGAGAAGAGTACC[FAM]G 86 hypoxia up-regulated 1 200825_s_at

NUCB2 NM_005013 TCACCTAAACCACCTGAATCCTGA CGGCGTTCCAGATCACTTGTTGC[FAM]G 87 nucleobindin 2 203675_at

procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline P4HB NM_000918 CGATTCACCAAGTACAAGCCCGAAT[FAM]G GGTGGCAGAACTCTGTGATCCTC 67 200656_s_at//1564494_s_at 4-hydroxylase), beta polypeptide

PDIA4 NM_004911 CGGGCAAGAACAACAAGGGACC[FAM]G CCATCACAATGGAGTCAAAGGTC 66 protein disulfide isomerase family A, member 4 208658_at//211048_s_at

SDF2L1 NM_022044 CGTATTCTCGCACGACATCAAATA[FAM]G GACGCCTCTACGCCGGTCAC 69 stromal cell-derived factor 2-like 1 218681_s_at

SEL1L NM_005065 CGTTTCAGGATATTCACCTTGCGAAA[FAM]G TGCAGAGGGCTAGGAAGACTGG 93 sel-1 suppressor of lin-12-like (C. elegans) 202061_s_at

TMED7 NM_181836 CGAGTTAGATAAAAGAACCACCACAACT[FAM]G GCAATGGTGCATCAATTCTCA 72 transmembrane emp24 protein transport domain containing 7 214658_at//209404_s_at

216449_x_at//200599_s_at HSP90B1 NM_003299 CGGAAGTTTGGTGTCGGTTTCTATTC[FAM]G TGTGCTGGGTATCGTTGTTGTG 88 heat shock protein 90kDa beta (Grp94), member 1 //200598_s_at//216450_x_at

UBE2J1 NM_016021 CGGAAGCATTGGCAGCTCTTATATTC[FAM]G CCCAATGCAGAATGTTTAATGAAG 133 ubiquitin-conjugating enzyme E2, J1 (UBC6 homolog, yeast) 217823_s_at//217826_s_at

ACTB Cat#_101H-01 (Invitrogen, Primer sequence is not available.) Actin, beta ņ

- 13 - Supplemental Table S5. Categorical analysis of the Glucose Deprivation signature by DAVID (2). Data shown are sorted by p-value.

A. Up-regulated 148 probe sets. (p < 0.001)

List Fold Category ID Term Count % PValue Genes FDR Total Enrichment 235341_at, 208499_s_at, 211936_at, 230031_at, 218681_s_at, 223299_at, 1554148_a_at, 203164_at, 203165_s_at, 220012_at, 231944_at, 203279_at, 230659_at, 200935_at, 214316_x_at, 225090_at, 223054_at, 218358_at, 221553_at, 1564494_s_at, 200656_s_at, 1554462_a_at, 202843_at, 202842_s_at, 214658_at, 223209_s_at, 207668_x_at, 208639_x_at, GOTERM_CC_ALL GO:0005783 endoplasmic reticulum 32 0.344 3.2E-22 216640_s_at, 217168_s_at, 200825_s_at, 200945_s_at, 208658_at, 211048_s_at, 202064_s_at, 202062_s_at, 202061_s_at, 69 8.89 5.0E-19 230265_at, 212902_at, 244841_at, 208612_at, 227033_at, 202558_s_at, 221781_s_at, 221782_at, 219600_s_at, 222907_x_at, 217823_s_at, 222435_s_at, 217826_s_at, 200598_s_at, 216449_x_at, 200599_s_at, 203675_at, 229838_at, 218696_at, 208959_s_at, 235341_at, 208499_s_at, 211936_at, 230031_at, 218681_s_at, 223299_at, 1554148_a_at, 203164_at, 203165_s_at, 220012_at, 231944_at, 203279_at, 230659_at, 200935_at, 214316_x_at, 218358_at, 1564494_s_at, 200656_s_at, SP_PIR_KEYWORDS ņ endoplasmic reticulum 25 0.269 3.4E-18 214658_at, 223209_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 217168_s_at, 200825_s_at, 200945_s_at, 202064_s_at, 75 10.36 5.4E-15 202062_s_at, 202061_s_at, 230265_at, 208658_at, 211048_s_at, 208612_at, 227033_at, 212902_at, 244841_at, 202558_s_at, 217823_s_at, 222435_s_at, 217826_s_at, 218696_at, 200598_s_at, 216449_x_at, 200599_s_at, 208959_s_at, 217168_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 200825_s_at, 211936_at, 230031_at, 200945_s_at, 208658_at, 211048_s_at, 202064_s_at, 202062_s_at, 202061_s_at, 230265_at, 1554148_a_at, 203164_at, 203165_s_at, 220012_at, GOTERM_CC_ALL GO:0044432 endoplasmic reticulum part 23 0.247 4.5E-17 231944_at, 208612_at, 227033_at, 203279_at, 230659_at, 225090_at, 200935_at, 214316_x_at, 221781_s_at, 221782_at, 69 10.70 7.0E-14 223054_at, 217823_s_at, 222435_s_at, 217826_s_at, 200598_s_at, 216449_x_at, 200599_s_at, 218696_at, 221553_at, 1564494_s_at, 200656_s_at, 214658_at, 208959_s_at, 223209_s_at, 200598_s_at, 216449_x_at, 200599_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 200825_s_at, 211936_at, 230031_at, GOTERM_CC_ALL GO:0005788 endoplasmic reticulum lumen 11 0.118 9.5E-14 208658_at, 211048_s_at, 208612_at, 227033_at, 1564494_s_at, 200656_s_at, 200935_at, 214316_x_at, 221781_s_at, 69 40.77 1.5E-10 221782_at, 208959_s_at, 223054_at, 200598_s_at, 216449_x_at, 200599_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 218681_s_at, 211936_at, 230031_at, INTERPRO IPR000886 Endoplasmic reticulum targeting sequence 9 0.097 3.6E-11 73 41.51 7.0E-08 208658_at, 211048_s_at, 1564494_s_at, 200656_s_at, 200935_at, 214316_x_at, 221781_s_at, 221782_at, 208959_s_at, short sequence motif:Prevents secretion 200598_s_at, 216449_x_at, 200599_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 218681_s_at, 211936_at, 230031_at, UP_SEQ_FEATURE ņ 8 0.086 1.5E-10 55 50.10 3.2E-07 from ER 208658_at, 211048_s_at, 208612_at, 227033_at, 1564494_s_at, 200656_s_at, 208959_s_at, 217168_s_at, 211936_at, 230031_at, 200945_s_at, 202064_s_at, 202062_s_at, 202061_s_at, 230265_at, 1554148_a_at, nuclear envelope-endoplasmic reticulum 203164_at, 203165_s_at, 220012_at, 231944_at, 203279_at, 230659_at, 225090_at, 217823_s_at, 222435_s_at, GOTERM_CC_ALL GO:0042175 16 0.172 4.6E-10 69 8.28 7.1E-07 network 217826_s_at, 200598_s_at, 216449_x_at, 200599_s_at, 203675_at, 229838_at, 218696_at, 221553_at, 214658_at, 208959_s_at, 223209_s_at, 217168_s_at, 211936_at, 230031_at, 200945_s_at, 202064_s_at, 202062_s_at, 202061_s_at, 230265_at, 1554148_a_at, GOTERM_CC_ALL GO:0005789 endoplasmic reticulum membrane 15 0.161 3.5E-09 203164_at, 203165_s_at, 220012_at, 231944_at, 203279_at, 230659_at, 225090_at, 217823_s_at, 222435_s_at, 69 7.92 5.5E-06 217826_s_at, 200598_s_at, 216449_x_at, 200599_s_at, 218696_at, 221553_at, 214658_at, 208959_s_at, 223209_s_at, 200598_s_at, 216449_x_at, 200599_s_at, 218696_at, 217168_s_at, 200825_s_at, 235341_at, 208499_s_at, 209383_at, GOTERM_BP_ALL GO:0051789 response to protein stimulus 9 0.097 5.5E-09 66 22.28 1.1E-05 203279_at, 230659_at, 208959_s_at, 223209_s_at, 200598_s_at, 216449_x_at, 200599_s_at, 218696_at, 217168_s_at, 200825_s_at, 235341_at, 208499_s_at, 209383_at, GOTERM_BP_ALL GO:0006986 response to unfolded protein 9 0.097 5.5E-09 66 22.28 1.1E-05 203279_at, 230659_at, 208959_s_at, 223209_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 209383_at, 208658_at, 211048_s_at, 208612_at, 227033_at, 1564494_s_at, GOTERM_BP_ALL GO:0045454 cell redox homeostasis 8 0.086 1.1E-08 66 28.21 2.1E-05 200656_s_at, 221781_s_at, 221782_at, 208959_s_at, 223209_s_at,

- 14 - 207668_x_at, 208639_x_at, 216640_s_at, 208658_at, 211048_s_at, 208612_at, 227033_at, 1564494_s_at, 200656_s_at, GOTERM_MF_ALL GO:0003756 protein disulfide isomerase activity 5 0.054 6.5E-08 74 114.65 1.2E-04 208959_s_at, 217168_s_at, 211936_at, 230031_at, 200945_s_at, 202064_s_at, 202062_s_at, 202061_s_at, 230265_at, 1554148_a_at, 203164_at, 203165_s_at, 212902_at, 244841_at, 220012_at, 231944_at, 203279_at, 230659_at, 225090_at, 217823_s_at, GOTERM_CC_ALL GO:0012505 endomembrane system 19 0.204 7.3E-08 69 4.55 1.1E-04 222435_s_at, 217826_s_at, 200598_s_at, 216449_x_at, 200599_s_at, 203675_at, 229838_at, 218696_at, 221553_at, 218871_x_at, 239077_at, 222235_s_at, 224999_at, 201984_s_at, 201983_s_at, 214658_at, 208959_s_at, 223209_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 208658_at, 211048_s_at, 208612_at, 227033_at, 1564494_s_at, 200656_s_at, INTERPRO IPR013766 Thioredoxin domain 6 0.065 1.2E-07 73 48.89 2.4E-04 221781_s_at, 221782_at, 208959_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 208658_at, 211048_s_at, 208612_at, 227033_at, 1564494_s_at, 200656_s_at, INTERPRO IPR006662 Thioredoxin-related 6 0.065 6.2E-07 73 35.77 1.2E-03 221781_s_at, 221782_at, 208959_s_at, 200598_s_at, 216449_x_at, 200599_s_at, 200825_s_at, 235341_at, 208499_s_at, 1564494_s_at, 200656_s_at, SP_PIR_KEYWORDS ņ chaperone 8 0.086 1.6E-06 75 13.91 2.5E-03 1554462_a_at, 202843_at, 202842_s_at, 200935_at, 214316_x_at, 208959_s_at, 223054_at, INTERPRO IPR005788 Disulphide isomerase 4 0.043 2.2E-06 207668_x_at, 208639_x_at, 216640_s_at, 208658_at, 211048_s_at, 208612_at, 227033_at, 1564494_s_at, 200656_s_at, 73 139.69 4.2E-03 200598_s_at, 216449_x_at, 200599_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 200825_s_at, 220012_at, 231944_at, GOTERM_BP_ALL GO:0006457 protein folding 10 0.108 2.3E-06 1564494_s_at, 200656_s_at, 1554462_a_at, 202843_at, 202842_s_at, 200935_at, 214316_x_at, 221781_s_at, 221782_at, 66 8.43 4.3E-03 208959_s_at, 223054_at, 218050_at, 209682_at, 227900_at, 223299_at, 209743_s_at, 209744_x_at, 203935_at, 220012_at, 231944_at, 203279_at, 230659_at, 209435_s_at, 200935_at, 214316_x_at, 225090_at, 223054_at, 220038_at, 218871_x_at, 239077_at, 222235_s_at, 221553_at, 224999_at, 201984_s_at, 201983_s_at, 1564494_s_at, 200656_s_at, 201000_at, 1554462_a_at, GOTERM_BP_ALL GO:0044267 cellular protein metabolic process 33 0.355 2.3E-06 202843_at, 202842_s_at, 223209_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 217168_s_at, 200825_s_at, 66 2.21 4.5E-03 1555788_a_at, 218145_at, 200802_at, 221781_s_at, 221782_at, 213532_at, 204203_at, 202129_s_at, 202131_s_at, 217823_s_at, 222435_s_at, 217826_s_at, 200598_s_at, 216449_x_at, 200599_s_at, 218696_at, 221050_s_at, 223789_s_at, 208959_s_at, SP_PIR_KEYWORDS ņ redox-active disulfide 4 0.043 2.5E-06 207668_x_at, 208639_x_at, 216640_s_at, 208658_at, 211048_s_at, 208612_at, 227033_at, 1564494_s_at, 200656_s_at, 75 134.09 3.9E-03 218050_at, 209682_at, 227900_at, 223299_at, 209743_s_at, 209744_x_at, 203935_at, 220012_at, 231944_at, 203279_at, 230659_at, 209435_s_at, 200935_at, 214316_x_at, 225090_at, 223054_at, 220038_at, 218871_x_at, 239077_at, 222235_s_at, 221553_at, 224999_at, 201984_s_at, 201983_s_at, 1564494_s_at, 200656_s_at, 201000_at, 1554462_a_at, GOTERM_BP_ALL GO:0044260 cellular macromolecule metabolic process 33 0.355 3.3E-06 202843_at, 202842_s_at, 223209_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 217168_s_at, 200825_s_at, 66 2.17 6.3E-03 1555788_a_at, 218145_at, 200802_at, 221781_s_at, 221782_at, 213532_at, 204203_at, 202129_s_at, 202131_s_at, 217823_s_at, 222435_s_at, 217826_s_at, 200598_s_at, 216449_x_at, 200599_s_at, 218696_at, 221050_s_at, 223789_s_at, 208959_s_at, UP_SEQ_FEATURE ņ domain:Thioredoxin 2 4 0.043 4.7E-06 207668_x_at, 208639_x_at, 216640_s_at, 208658_at, 211048_s_at, 208612_at, 227033_at, 1564494_s_at, 200656_s_at, 55 109.60 1.0E-02 UP_SEQ_FEATURE ņ domain:Thioredoxin 1 4 0.043 4.7E-06 207668_x_at, 208639_x_at, 216640_s_at, 208658_at, 211048_s_at, 208612_at, 227033_at, 1564494_s_at, 200656_s_at, 55 109.60 1.0E-02 207668_x_at, 208639_x_at, 216640_s_at, 208658_at, 211048_s_at, 221553_at, 208612_at, 227033_at, 1564494_s_at, INTERPRO IPR012335 Thioredoxin fold 7 0.075 5.4E-06 73 15.56 1.0E-02 200656_s_at, 221781_s_at, 221782_at, 208959_s_at, PIR_SUPERFAMILY PIRSF001487 protein disulfide-isomerase 4 0.043 6.0E-06 207668_x_at, 208639_x_at, 216640_s_at, 208612_at, 227033_at, 1564494_s_at, 200656_s_at, 208959_s_at, 35 98.84 1.1E-02 200598_s_at, 216449_x_at, 200599_s_at, 218696_at, 217168_s_at, 200825_s_at, 235341_at, 208499_s_at, 209383_at, GOTERM_BP_ALL GO:0009607 response to biotic stimulus 10 0.108 6.3E-06 66 7.44 1.2E-02 211936_at, 230031_at, 203279_at, 230659_at, 208959_s_at, 223209_s_at, 200598_s_at, 216449_x_at, 200599_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 209383_at, 208658_at, 211048_s_at, GOTERM_BP_ALL GO:0019725 cellular homeostasis 10 0.108 6.3E-06 208612_at, 227033_at, 1564494_s_at, 200656_s_at, 200935_at, 214316_x_at, 221781_s_at, 221782_at, 208959_s_at, 66 7.44 1.2E-02 223209_s_at, 217168_s_at, 235341_at, 208499_s_at, 200825_s_at, 211936_at, 230031_at, 209383_at, 209743_s_at, 209744_x_at, GOTERM_BP_ALL GO:0006950 response to stress 17 0.183 7.9E-06 203935_at, 1555788_a_at, 218145_at, 203279_at, 230659_at, 225090_at, 204203_at, 220038_at, 200598_s_at, 66 3.66 1.5E-02 216449_x_at, 200599_s_at, 218696_at, 224999_at, 201984_s_at, 201983_s_at, 208959_s_at, 223209_s_at,

- 15 - 218050_at, 209682_at, 227900_at, 223299_at, 209743_s_at, 209744_x_at, 203935_at, 220012_at, 231944_at, 203279_at, 230659_at, 209435_s_at, 200935_at, 214316_x_at, 225090_at, 223054_at, 220038_at, 218871_x_at, 239077_at, 222235_s_at, 221553_at, 224999_at, 201984_s_at, 201983_s_at, 1564494_s_at, 200656_s_at, 201000_at, 1554462_a_at, GOTERM_BP_ALL GO:0019538 protein metabolic process 33 0.355 8.8E-06 202843_at, 202842_s_at, 223209_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 217168_s_at, 200825_s_at, 66 2.08 1.7E-02 1555788_a_at, 218145_at, 200802_at, 221781_s_at, 221782_at, 213532_at, 204203_at, 202129_s_at, 202131_s_at, 217823_s_at, 222435_s_at, 217826_s_at, 200598_s_at, 216449_x_at, 200599_s_at, 218696_at, 221050_s_at, 223789_s_at, 208959_s_at, 200598_s_at, 216449_x_at, 200599_s_at, 211936_at, 230031_at, 220012_at, 231944_at, 1554462_a_at, 202843_at, GOTERM_MF_ALL GO:0051082 unfolded protein binding 7 0.075 2.4E-05 74 11.98 4.3E-02 202842_s_at, 200935_at, 214316_x_at, 221781_s_at, 221782_at, 223054_at, 200598_s_at, 216449_x_at, 200599_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 208658_at, 211048_s_at, 208612_at, GOTERM_CC_ALL GO:0048770 pigment granule 6 0.065 3.0E-05 69 16.42 4.6E-02 227033_at, 1564494_s_at, 200656_s_at, 209610_s_at, 212811_x_at, 212810_s_at, 200598_s_at, 216449_x_at, 200599_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 208658_at, 211048_s_at, 208612_at, GOTERM_CC_ALL GO:0042470 melanosome 6 0.065 3.0E-05 69 16.42 4.6E-02 227033_at, 1564494_s_at, 200656_s_at, 209610_s_at, 212811_x_at, 212810_s_at, SP_PIR_KEYWORDS ņ unfolded protein response 4 0.043 3.1E-05 218696_at, 217168_s_at, 235341_at, 208499_s_at, 203279_at, 230659_at, 75 62.57 5.0E-02 207668_x_at, 208639_x_at, 216640_s_at, 208658_at, 211048_s_at, 220012_at, 231944_at, 208612_at, 227033_at, SP_PIR_KEYWORDS ņ redox-active center 5 0.054 3.4E-05 75 26.67 5.5E-02 1564494_s_at, 200656_s_at, 203675_at, 229838_at, 207668_x_at, 208639_x_at, 216640_s_at, 211936_at, 230031_at, 1564494_s_at, 200656_s_at, GOTERM_CC_ALL GO:0005793 ER-Golgi intermediate compartment 5 0.054 3.7E-05 69 26.11 5.7E-02 208959_s_at, 200598_s_at, 216449_x_at, 200599_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 209383_at, 208658_at, 211048_s_at, GOTERM_BP_ALL GO:0042592 homeostatic process 10 0.108 6.2E-05 208612_at, 227033_at, 1564494_s_at, 200656_s_at, 200935_at, 214316_x_at, 221781_s_at, 221782_at, 208959_s_at, 66 5.57 1.2E-01 223209_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 208658_at, 211048_s_at, 208612_at, 227033_at, 1564494_s_at, 200656_s_at, GOTERM_MF_ALL GO:0016860 intramolecular oxidoreductase activity 5 0.054 6.8E-05 74 22.48 1.2E-01 208959_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 208658_at, 211048_s_at, 220012_at, 231944_at, 208612_at, 227033_at, GOTERM_MF_ALL GO:0009055 electron carrier activity 8 0.086 7.3E-05 74 7.71 1.3E-01 224999_at, 201984_s_at, 201983_s_at, 1564494_s_at, 200656_s_at, 221781_s_at, 221782_at, 208959_s_at, 211936_at, 230031_at, 1554148_a_at, 203164_at, 203165_s_at, 220012_at, 231944_at, 203279_at, 230659_at, 209435_s_at, 200935_at, 214316_x_at, 225090_at, 223054_at, 229453_at, 218871_x_at, 239077_at, 222235_s_at, 221553_at, 224999_at, 201984_s_at, 201983_s_at, 1564494_s_at, 200656_s_at, 1554462_a_at, 202843_at, 202842_s_at, GOTERM_CC_ALL GO:0044446 intracellular organelle part 30 0.323 1.1E-04 214658_at, 223209_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 217168_s_at, 200825_s_at, 200945_s_at, 202064_s_at, 69 1.97 1.8E-01 202062_s_at, 202061_s_at, 230265_at, 208658_at, 211048_s_at, 212902_at, 244841_at, 208612_at, 227033_at, 221781_s_at, 221782_at, 217823_s_at, 222435_s_at, 217826_s_at, 200598_s_at, 216449_x_at, 200599_s_at, 203675_at, 229838_at, 218696_at, 208959_s_at, site:Lowers pKa of C-terminal Cys of UP_SEQ_FEATURE ņ 3 0.032 1.2E-04 207668_x_at, 208639_x_at, 216640_s_at, 208612_at, 227033_at, 1564494_s_at, 200656_s_at, 55 164.40 2.5E-01 second active site site:Lowers pKa of C-terminal Cys of first UP_SEQ_FEATURE ņ 3 0.032 1.2E-04 207668_x_at, 208639_x_at, 216640_s_at, 208612_at, 227033_at, 1564494_s_at, 200656_s_at, 55 164.40 2.5E-01 active site GOTERM_BP_ALL GO:0006984 ER-nuclear signaling pathway 4 0.043 1.2E-04 218696_at, 209383_at, 211936_at, 230031_at, 223209_s_at, 66 40.47 2.3E-01 211936_at, 230031_at, 1554148_a_at, 203164_at, 203165_s_at, 220012_at, 231944_at, 203279_at, 230659_at, 209435_s_at, 200935_at, 214316_x_at, 225090_at, 223054_at, 229453_at, 218871_x_at, 239077_at, 222235_s_at, 221553_at, 224999_at, 201984_s_at, 201983_s_at, 1564494_s_at, 200656_s_at, 1554462_a_at, 202843_at, 202842_s_at, GOTERM_CC_ALL GO:0044422 organelle part 30 0.323 1.2E-04 214658_at, 223209_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 217168_s_at, 200825_s_at, 200945_s_at, 202064_s_at, 69 1.96 1.9E-01 202062_s_at, 202061_s_at, 230265_at, 208658_at, 211048_s_at, 212902_at, 244841_at, 208612_at, 227033_at, 221781_s_at, 221782_at, 217823_s_at, 222435_s_at, 217826_s_at, 200598_s_at, 216449_x_at, 200599_s_at, 203675_at, 229838_at, 218696_at, 208959_s_at, INTERPRO IPR005792 Protein disulphide isomerase 3 0.032 1.6E-04 208658_at, 211048_s_at, 208612_at, 227033_at, 1564494_s_at, 200656_s_at, 73 146.67 3.1E-01 GOTERM_BP_ALL GO:0006983 ER overload response 3 0.032 1.7E-04 218696_at, 209383_at, 211936_at, 230031_at, 66 139.64 3.3E-01

- 16 - 200598_s_at, 216449_x_at, 200599_s_at, 209238_at, 216985_s_at, 209682_at, 227900_at, 200945_s_at, 208658_at, GOTERM_BP_ALL GO:0045184 establishment of protein localization 12 0.129 2.6E-04 211048_s_at, 229618_at, 212902_at, 244841_at, 208612_at, 227033_at, 224999_at, 201984_s_at, 201983_s_at, 66 3.75 5.0E-01 209435_s_at, 200935_at, 214316_x_at, 223209_s_at, UP_SEQ_FEATURE ņ site:Contributes to redox potential value 3 0.032 2.9E-04 207668_x_at, 208639_x_at, 216640_s_at, 208612_at, 227033_at, 1564494_s_at, 200656_s_at, 55 109.60 6.3E-01 200598_s_at, 216449_x_at, 200599_s_at, 209238_at, 216985_s_at, 209682_at, 227900_at, 200945_s_at, 208658_at, GOTERM_BP_ALL GO:0008104 protein localization 12 0.129 4.4E-04 211048_s_at, 229618_at, 212902_at, 244841_at, 208612_at, 227033_at, 224999_at, 201984_s_at, 201983_s_at, 66 3.54 8.3E-01 209435_s_at, 200935_at, 214316_x_at, 223209_s_at, GOTERM_BP_ALL GO:0030503 regulation of cell redox homeostasis 3 0.032 4.9E-04 209383_at, 208959_s_at, 223209_s_at, 66 87.27 9.3E-01 207668_x_at, 208639_x_at, 216640_s_at, 200825_s_at, 211936_at, 230031_at, 208658_at, 211048_s_at, 208612_at, GOTERM_CC_ALL GO:0031974 membrane-enclosed lumen 13 0.140 5.6E-04 227033_at, 200935_at, 214316_x_at, 221781_s_at, 221782_at, 223054_at, 200598_s_at, 216449_x_at, 200599_s_at, 69 3.20 8.6E-01 229453_at, 1564494_s_at, 200656_s_at, 1554462_a_at, 202843_at, 202842_s_at, 208959_s_at, 207668_x_at, 208639_x_at, 216640_s_at, 200825_s_at, 211936_at, 230031_at, 208658_at, 211048_s_at, 208612_at, GOTERM_CC_ALL GO:0043233 organelle lumen 13 0.140 5.6E-04 227033_at, 200935_at, 214316_x_at, 221781_s_at, 221782_at, 223054_at, 200598_s_at, 216449_x_at, 200599_s_at, 69 3.20 8.6E-01 229453_at, 1564494_s_at, 200656_s_at, 1554462_a_at, 202843_at, 202842_s_at, 208959_s_at, 221577_x_at, 218681_s_at, 211936_at, 230031_at, 203935_at, 220012_at, 231944_at, 200935_at, 214316_x_at, 202655_at, 209270_at, 218358_at, 224999_at, 201984_s_at, 201983_s_at, 1564494_s_at, 200656_s_at, 214658_at, SP_PIR_KEYWORDS ņ signal 25 0.269 6.0E-04 207668_x_at, 208639_x_at, 216640_s_at, 200825_s_at, 202064_s_at, 202062_s_at, 202061_s_at, 230265_at, 208658_at, 75 2.01 9.5E-01 211048_s_at, 208612_at, 227033_at, 202558_s_at, 213532_at, 221781_s_at, 221782_at, 218696_at, 203675_at, 229838_at, 200598_s_at, 216449_x_at, 200599_s_at, 209099_x_at, 216268_s_at, 208959_s_at, 200598_s_at, 216449_x_at, 200599_s_at, 209238_at, 216985_s_at, 209682_at, 227900_at, 200945_s_at, 208658_at, GOTERM_BP_ALL GO:0033036 macromolecule localization 12 0.129 7.3E-04 211048_s_at, 229618_at, 212902_at, 244841_at, 208612_at, 227033_at, 224999_at, 201984_s_at, 201983_s_at, 66 3.32 1.4E+00 209435_s_at, 200935_at, 214316_x_at, 223209_s_at, Molecular chaperone, heat shock protein, INTERPRO IPR015609 4 0.043 7.4E-04 235341_at, 208499_s_at, 1554462_a_at, 202843_at, 202842_s_at, 221781_s_at, 221782_at, 223054_at, 73 22.22 1.4E+00 Hsp40, Dn

B. Down-regulated 98 probe sets. (p < 0.001)

List Fold Category ID Term Count % PValue Genes FDR Total Enrichment 202240_at, 228045_at, 221520_s_at, 215942_s_at, 201969_at, 242300_at, 204603_at, 232238_at, 219918_s_at, 239002_at, 203625_x_at, 203626_s_at, 210567_s_at, 235572_at, 209891_at, 1555758_a_at, 209714_s_at, 204827_s_at, 208079_s_at, GOTERM_BP_ALL GO:0007049 cell cycle 29 0.341 2.4E-18 202094_at, 215731_s_at, 221965_at, 227896_at, 242300_at, 219990_at, 203418_at, 213226_at, 221436_s_at, 223307_at, 65 7.67 4.6E-15 204444_at, 205436_s_at, 215629_s_at, 216870_x_at, 220085_at, 242890_at, 227350_at, 227349_at, 211814_s_at, 204159_at, 203145_at, 212020_s_at, 212023_s_at, 202094_at, 215731_s_at, 221965_at, 202240_at, 221520_s_at, 228045_at, 215942_s_at, 203418_at, 213226_at, 204444_at, 203625_x_at, 203626_s_at, 210567_s_at, 232238_at, 219918_s_at, 239002_at, 204603_at, 221436_s_at, 223307_at, GOTERM_BP_ALL GO:0022403 cell cycle phase 21 0.247 8.7E-18 65 13.94 1.7E-14 205436_s_at, 235572_at, 209891_at, 220085_at, 242890_at, 227350_at, 227349_at, 204159_at, 1555758_a_at, 209714_s_at, 203145_at, 204827_s_at, 208079_s_at, 202094_at, 215731_s_at, 221965_at, 202240_at, 228045_at, 221520_s_at, 215942_s_at, 203418_at, 213226_at, 204444_at, 203625_x_at, 203626_s_at, 210567_s_at, 232238_at, 219918_s_at, 239002_at, 221436_s_at, 223307_at, 235572_at, GOTERM_BP_ALL GO:0000278 mitotic cell cycle 19 0.224 4.4E-16 65 14.12 8.5E-13 209891_at, 220085_at, 242890_at, 227350_at, 227349_at, 204159_at, 1555758_a_at, 209714_s_at, 203145_at, 204827_s_at, 208079_s_at, 202240_at, 228045_at, 221520_s_at, 215942_s_at, 204603_at, 232238_at, 219918_s_at, 239002_at, 203625_x_at, 203626_s_at, 210567_s_at, 235572_at, 209891_at, 1555758_a_at, 209714_s_at, 204827_s_at, 208079_s_at, 202094_at, GOTERM_BP_ALL GO:0022402 cell cycle process 25 0.294 9.4E-16 65 7.89 1.7E-12 215731_s_at, 221965_at, 227896_at, 203418_at, 213226_at, 221436_s_at, 223307_at, 204444_at, 205436_s_at, 215629_s_at, 216870_x_at, 220085_at, 242890_at, 227350_at, 227349_at, 204159_at, 211814_s_at, 203145_at,

- 17 - 212020_s_at, 212023_s_at,

202094_at, 215731_s_at, 221965_at, 202240_at, 228045_at, 221520_s_at, 215942_s_at, 203418_at, 213226_at, 204444_at, GOTERM_BP_ALL GO:0000279 M phase 18 0.212 1.6E-15 232238_at, 219918_s_at, 239002_at, 204603_at, 221436_s_at, 223307_at, 205436_s_at, 235572_at, 209891_at, 220085_at, 65 14.82 3.2E-12 242890_at, 227350_at, 227349_at, 203145_at, 204827_s_at, 208079_s_at, 202094_at, 221520_s_at, 201969_at, 203418_at, 213226_at, 204444_at, 232238_at, 219918_s_at, 239002_at, 221436_s_at, SP_PIR_KEYWORDS ņ cell cycle 19 0.224 1.0E-14 223307_at, 205436_s_at, 215629_s_at, 216870_x_at, 235572_at, 209891_at, 220085_at, 242890_at, 227350_at, 227349_at, 68 11.94 1.6E-11 211814_s_at, 204159_at, 1555758_a_at, 209714_s_at, 203145_at, 204827_s_at, 212020_s_at, 212023_s_at, 208079_s_at, 202094_at, 215731_s_at, 221965_at, 202240_at, 228045_at, 221520_s_at, 215942_s_at, 203418_at, 213226_at, 204444_at, GOTERM_BP_ALL GO:0000087 M phase of mitotic cell cycle 16 0.188 2.1E-14 232238_at, 219918_s_at, 239002_at, 221436_s_at, 223307_at, 235572_at, 209891_at, 220085_at, 242890_at, 227350_at, 65 16.51 4.0E-11 227349_at, 203145_at, 204827_s_at, 208079_s_at, 202094_at, 202240_at, 228045_at, 221520_s_at, 215942_s_at, 203418_at, 213226_at, 204444_at, 232238_at, 219918_s_at, GOTERM_BP_ALL GO:0007067 mitosis 15 0.177 4.0E-13 239002_at, 221436_s_at, 223307_at, 235572_at, 209891_at, 220085_at, 242890_at, 227350_at, 227349_at, 203145_at, 65 15.62 7.6E-10 204827_s_at, 208079_s_at, 218115_at, 202240_at, 229305_at, 221520_s_at, 228045_at, 215942_s_at, 218755_at, 200988_s_at, 201969_at, 201890_at, 209773_s_at, 242300_at, 201038_s_at, 212170_at, 229129_at, 203625_x_at, 203626_s_at, 210567_s_at, 204603_at, 232238_at, 219918_s_at, 239002_at, 207165_at, 209709_s_at, 235572_at, 1555758_a_at, 209714_s_at, 204067_at, SP_PIR_KEYWORDS ņ phosphoprotein 47 0.553 8.9E-13 208079_s_at, 201316_at, 227578_at, 202094_at, 208955_at, 215731_s_at, 221965_at, 213449_at, 225687_at, 225108_at, 68 2.59 1.4E-09 209377_s_at, 211042_x_at, 213852_at, 217856_at, 200685_at, 201129_at, 204444_at, 221436_s_at, 223307_at, 203276_at, 205436_s_at, 211814_s_at, 221521_s_at, 227164_at, 203145_at, 220651_s_at, 222962_s_at, 223570_at, 212020_s_at, 212023_s_at, 238529_at, 221727_at, 202094_at, 204444_at, 221436_s_at, 223307_at, 232238_at, 219918_s_at, 239002_at, 221520_s_at, 235572_at, 220085_at, SP_PIR_KEYWORDS ņ mitosis 11 0.129 5.1E-11 68 22.24 8.1E-08 242890_at, 227350_at, 227349_at, 209891_at, 203145_at, 204827_s_at, 203418_at, 213226_at, 218115_at, 202240_at, 229305_at, 224428_s_at, 221520_s_at, 200988_s_at, 201969_at, 242300_at, 201038_s_at, 212170_at, 229129_at, 204603_at, 232238_at, 219918_s_at, 239002_at, 235572_at, 209891_at, 204827_s_at, 201316_at, 227578_at, 202094_at, 208955_at, 213449_at, 209377_s_at, 213852_at, 217856_at, 208937_s_at, 200685_at, 201129_at, SP_PIR_KEYWORDS ņ nucleus 39 0.459 1.9E-10 68 2.72 3.0E-07 203276_at, 205436_s_at, 220085_at, 242890_at, 227350_at, 227349_at, 218866_s_at, 211814_s_at, 221521_s_at, 203744_at, 227164_at, 220651_s_at, 222962_s_at, 223570_at, 212020_s_at, 212023_s_at, 238529_at, 221727_at, 205909_at, 202094_at, 204444_at, 221436_s_at, 223307_at, 232238_at, 219918_s_at, 239002_at, 221520_s_at, 235572_at, 220085_at, SP_PIR_KEYWORDS ņ cell division 12 0.141 2.3E-10 68 15.53 3.6E-07 242890_at, 227350_at, 227349_at, 209891_at, 211814_s_at, 203145_at, 204827_s_at, 203418_at, 213226_at, 202094_at, 218115_at, 229305_at, 228045_at, 221520_s_at, 209377_s_at, 229129_at, 205436_s_at, 235572_at, 209891_at, GOTERM_CC_ALL GO:0044427 chromosomal part 14 0.165 1.0E-09 69 9.87 1.6E-06 220085_at, 242890_at, 227350_at, 227349_at, 203744_at, 238529_at, 227578_at, 218115_at, 202240_at, 229305_at, 224428_s_at, 221520_s_at, 201969_at, 242300_at, 201038_s_at, 212170_at, 229129_at, 204603_at, 232238_at, 219918_s_at, 239002_at, 235572_at, 209891_at, 204827_s_at, 208079_s_at, 201316_at, 227578_at, 202094_at, 208955_at, 213449_at, 227896_at, 242300_at, 209377_s_at, 219990_at, 203418_at, 213226_at, 213852_at, GOTERM_CC_ALL GO:0005634 nucleus 45 0.529 1.7E-09 69 2.22 2.6E-06 217856_at, 208937_s_at, 200685_at, 201129_at, 203276_at, 218726_at, 205436_s_at, 220085_at, 242890_at, 227350_at, 227349_at, 218866_s_at, 211814_s_at, 204159_at, 221521_s_at, 203744_at, 227164_at, 220651_s_at, 222962_s_at, 223570_at, 212020_s_at, 212023_s_at, 238529_at, 221727_at, 205909_at, 202094_at, 204444_at, 221436_s_at, 223307_at, 232238_at, 219918_s_at, 239002_at, 221520_s_at, 235572_at, 220085_at, GOTERM_BP_ALL GO:0051301 cell division 12 0.141 4.8E-09 65 11.57 9.2E-06 242890_at, 227350_at, 227349_at, 209891_at, 211814_s_at, 203145_at, 204827_s_at, 203418_at, 213226_at, 202094_at, 218115_at, 229305_at, 228045_at, 221520_s_at, 209377_s_at, 229129_at, 205436_s_at, 235572_at, 209891_at, GOTERM_CC_ALL GO:0005694 chromosome 14 0.165 7.8E-09 69 8.36 1.2E-05 220085_at, 242890_at, 227350_at, 227349_at, 203744_at, 238529_at, 227578_at, 202094_at, 215731_s_at, 221965_at, 202240_at, 227896_at, 228045_at, 215942_s_at, 203418_at, 213226_at, 203625_x_at, GOTERM_BP_ALL GO:0000074 regulation of progression through cell cycle 14 0.165 2.0E-07 203626_s_at, 210567_s_at, 215629_s_at, 216870_x_at, 204159_at, 211814_s_at, 1555758_a_at, 209714_s_at, 65 6.29 3.9E-04 204827_s_at, 212020_s_at, 212023_s_at,

- 18 - 202094_at, 215731_s_at, 221965_at, 202240_at, 227896_at, 228045_at, 215942_s_at, 203418_at, 213226_at, 203625_x_at, GOTERM_BP_ALL GO:0051726 regulation of cell cycle 14 0.165 2.2E-07 203626_s_at, 210567_s_at, 215629_s_at, 216870_x_at, 204159_at, 211814_s_at, 1555758_a_at, 209714_s_at, 65 6.25 4.2E-04 204827_s_at, 212020_s_at, 212023_s_at, SP_PIR_KEYWORDS ņ Chromosomal protein 8 0.094 4.0E-07 202094_at, 229129_at, 229305_at, 221520_s_at, 205436_s_at, 203744_at, 238529_at, 227578_at, 68 16.97 6.3E-04

GOTERM_CC_ALL GO:0000775 chromosome, pericentric region 7 0.082 6.7E-07 202094_at, 229305_at, 221520_s_at, 228045_at, 235572_at, 220085_at, 242890_at, 227350_at, 227349_at, 209891_at, 69 22.04 1.0E-03

242300_at, 218115_at, 229305_at, 202240_at, 221520_s_at, 228045_at, 215942_s_at, 218755_at, 242300_at, 229129_at, intracellular non-membrane-bound GOTERM_CC_ALL GO:0043232 25 0.294 8.7E-07 235572_at, 209891_at, 208079_s_at, 227578_at, 202094_at, 213449_at, 209377_s_at, 203276_at, 204444_at, 205436_s_at, 69 2.94 1.4E-03 organelle 220085_at, 242890_at, 227350_at, 227349_at, 203744_at, 203145_at, 238529_at, 212020_s_at, 212023_s_at, 242300_at, 218115_at, 229305_at, 202240_at, 221520_s_at, 228045_at, 215942_s_at, 218755_at, 242300_at, 229129_at, GOTERM_CC_ALL GO:0043228 non-membrane-bound organelle 25 0.294 8.7E-07 235572_at, 209891_at, 208079_s_at, 227578_at, 202094_at, 213449_at, 209377_s_at, 203276_at, 204444_at, 205436_s_at, 69 2.94 1.4E-03 220085_at, 242890_at, 227350_at, 227349_at, 203744_at, 203145_at, 238529_at, 212020_s_at, 212023_s_at, SP_PIR_KEYWORDS ņ centromere 5 0.059 8.2E-06 202094_at, 229305_at, 221520_s_at, 235572_at, 209891_at, 68 38.06 1.3E-02 203625_x_at, 203626_s_at, 210567_s_at, 204159_at, 1555758_a_at, 209714_s_at, 212020_s_at, 212023_s_at, 203418_at, SP_PIR_KEYWORDS ņ cell cycle control 5 0.059 1.2E-05 68 34.97 1.8E-02 213226_at, 242300_at, 218115_at, 202240_at, 229305_at, 224428_s_at, 228045_at, 201890_at, 209773_s_at, 201969_at, 242300_at, 242300_at, 229129_at, 203625_x_at, 203626_s_at, 210567_s_at, 204603_at, 215030_at, 1555758_a_at, 209714_s_at, 208079_s_at, 201316_at, 208955_at, 213449_at, 227896_at, 242300_at, 219990_at, 214331_at, 213852_at, 217856_at, GOTERM_BP_ALL GO:0043283 biopolymer metabolic process 39 0.459 5.4E-05 65 1.72 1.0E-01 208937_s_at, 200685_at, 201129_at, 221436_s_at, 223307_at, 205436_s_at, 220085_at, 242890_at, 227350_at, 227349_at, 218866_s_at, 211814_s_at, 221521_s_at, 203744_at, 227164_at, 220651_s_at, 222962_s_at, 223570_at, 238529_at, 221727_at, 205909_at, regulation of cyclin-dependent protein GOTERM_BP_ALL GO:0000079 5 0.059 5.9E-05 227896_at, 211814_s_at, 204159_at, 1555758_a_at, 209714_s_at, 203418_at, 213226_at, 65 23.17 1.1E-01 kinase acti 208955_at, 204603_at, 211814_s_at, 221521_s_at, 201969_at, 201890_at, 209773_s_at, 220651_s_at, 222962_s_at, GOTERM_BP_ALL GO:0006260 DNA replication 8 0.094 7.8E-05 65 7.56 1.5E-01 223570_at, 205909_at, INTERPRO IPR000504 RNA recognition motif, RNP-1 7 0.082 9.4E-05 229129_at, 201129_at, 215030_at, 227164_at, 213852_at, 217856_at, 200685_at, 212170_at, 62 9.33 1.8E-01 INTERPRO IPR012677 Nucleotide-binding, alpha-beta plait 7 0.082 1.0E-04 229129_at, 201129_at, 215030_at, 227164_at, 213852_at, 217856_at, 200685_at, 212170_at, 62 9.16 2.0E-01 SP_PIR_KEYWORDS ņ cyclin 5 0.059 1.3E-04 211814_s_at, 204159_at, 1555758_a_at, 209714_s_at, 204827_s_at, 203418_at, 213226_at, 68 19.03 2.1E-01

GOTERM_CC_ALL GO:0000785 chromatin 7 0.082 1.8E-04 218115_at, 205436_s_at, 220085_at, 242890_at, 227350_at, 227349_at, 209377_s_at, 203744_at, 238529_at, 227578_at, 69 8.29 2.8E-01

208955_at, 218115_at, 227896_at, 201890_at, 209773_s_at, 201969_at, 204603_at, 205436_s_at, 220085_at, 242890_at, GOTERM_BP_ALL GO:0006259 DNA metabolic process 13 0.153 1.9E-04 65 3.57 3.7E-01 227350_at, 227349_at, 211814_s_at, 221521_s_at, 238529_at, 220651_s_at, 222962_s_at, 223570_at, 205909_at, SMART SM00360 RRM 7 0.082 2.2E-04 229129_at, 201129_at, 215030_at, 227164_at, 213852_at, 217856_at, 200685_at, 212170_at, 39 7.72 3.2E-01 SP_PIR_KEYWORDS ņ dna replication 5 0.059 2.6E-04 221521_s_at, 201969_at, 201890_at, 209773_s_at, 220651_s_at, 222962_s_at, 223570_at, 205909_at, 68 15.98 4.1E-01 218115_at, 229305_at, 224428_s_at, 201890_at, 209773_s_at, 201969_at, 242300_at, 229129_at, 204603_at, 215030_at, 227578_at, 208955_at, 227896_at, 213449_at, 242300_at, 219990_at, 203418_at, 213226_at, 213852_at, 217856_at, nucleobase, nucleoside, nucleotide and GOTERM_BP_ALL GO:0006139 31 0.365 3.3E-04 208937_s_at, 200685_at, 201129_at, 205436_s_at, 220085_at, 242890_at, 227350_at, 227349_at, 218866_s_at, 65 1.81 6.3E-01 nucleic aci 211814_s_at, 221521_s_at, 203744_at, 227164_at, 220651_s_at, 222962_s_at, 223570_at, 238529_at, 221727_at, 205909_at, GOTERM_BP_ALL GO:0051329 interphase of mitotic cell cycle 5 0.059 5.8E-04 202094_at, 203625_x_at, 203626_s_at, 210567_s_at, 215942_s_at, 204159_at, 1555758_a_at, 209714_s_at, 65 12.84 1.1E+00 GOTERM_BP_ALL GO:0051325 interphase 5 0.059 7.4E-04 202094_at, 203625_x_at, 203626_s_at, 210567_s_at, 215942_s_at, 204159_at, 1555758_a_at, 209714_s_at, 65 12.06 1.4E+00

- 19 - Supplemental Table S6. List of the VST/biguanide signature genes. The annotation data was obtained from NetAffyx (Affymetrix). Data shown are sorted by Score (d) from SAM analysis.

A. Up-regulated 10 probe sets (9 genes) Gene Score Fold q-value Probe Set ID Gene Title Symbol (d) Change (%) 209383_at DDIT3 DNA-damage-inducible transcript 3 5.497 3.45 19.134 218651_s_at LARP6 La ribonucleoprotein domain family, member 6 5.355 3.03 19.134 225956_at C5orf41 chromosome 5 open reading frame 41 4.762 2.25 19.134 211527_x_at VEGFA vascular endothelial growth factor A 4.346 2.60 19.134 solute carrier family 1 (glutamate/neutral amino acid 212811_x_at SLC1A4 4.322 3.07 19.134 transporter), member 4 204203_at CEBPG CCAAT/enhancer binding protein (C/EBP), gamma 4.108 2.22 19.302 218145_at TRIB3 tribbles homolog 3 (Drosophila) 4.065 2.62 19.302 1555788_a_at TRIB3 tribbles homolog 3 (Drosophila) 3.983 2.73 19.302 226537_at HINT3 histidine triad nucleotide binding protein 3 3.949 2.38 19.302 209230_s_at NUPR1 nuclear protein 1 3.801 3.10 19.302

B. Down-regulated 15 probe sets (15 genes) Gene Score Fold q-value Probe Set ID Gene Title Symbol (d) Change (%) 242890_at --- CDNA FLJ34225 fis, clone FCBBF3023372 -7.372 0.52 0 212847_at FUBP1 Far upstream element (FUSE) binding protein 1 -6.866 0.54 0 226934_at CPSF6 cleavage and polyadenylation specific factor 6, 68kDa -5.945 0.57 0 proteasome (prosome, macropain) 26S subunit, ATPase, 201067_at PSMC2 -5.679 0.59 0 2 228694_at --- Homo sapiens, clone IMAGE:3352913, mRNA -5.154 0.60 13.667 228039_at DDX46 DEAD (Asp-Glu-Ala-Asp) box polypeptide 46 -5.102 0.58 13.667 processing of precursor 1, ribonuclease P/MRP subunit 213449_at POP1 -4.948 0.55 13.667 (S. cerevisiae) 227896_at BCCIP BRCA2 and CDKN1A interacting protein -4.536 0.50 13.667 227095_at --- CDNA: FLJ22522 fis, clone HRC12491 -4.364 0.53 19.134 1557487_at --- CDNA clone IMAGE:4797099 -4.320 0.58 19.134 221965_at MPHOSPH9 M-phase phosphoprotein 9 -4.171 0.55 19.134 226432_at ETNK1 ethanolamine kinase 1 -4.110 0.54 19.134 solute carrier family 7 (cationic amino acid transporter, SLC7A6 /// 203579_s_at y+ system), member 6 /// transient receptor potential -4.049 0.57 19.134 TRPV6 cation channel, subfamily V, member 6 231576_at --- Transcribed locus -4.001 0.53 19.134 204317_at GTSE1 G-2 and S-phase expressed 1 -3.992 0.54 19.134

- 20 - Supplemental Table S7. Pattern-matching analysis with the VST/biguanide signature. The pattern-matching analysis with the VST/biguanide signature genes was performed using the Connectivity Map database build 02 (3). Data shown are sorted by rank.

A. Connected perturbagens and cell line (Top10) Percent rank cmap name and cell line mean n enrichment p specificity non-null 1 pyrvinium - MCF7 0.927 4 0.990 0 0.0046 100 2 thioridazine - PC3 0.803 5 0.964 0 0.0000 100 3 0317956-0000 - MCF7 -0.742 4 -0.956 0 0.0066 100 4 niclosamide - MCF7 0.966 2 0.998 2.0E-05 0.0000 100 5 5224221 - MCF7 0.951 2 0.996 2.0E-05 0.0112 100 6 resveratrol - MCF7 0.656 6 0.855 2.0E-05 0.0316 100 7 terfenadine - MCF7 0.903 2 0.993 4.0E-05 0.0052 100 8 suloctidil - MCF7 0.905 2 0.993 4.0E-05 0.0000 100 9 valinomycin - MCF7 0.922 2 0.993 4.0E-05 0.0000 100 10 rottlerin - MCF7 0.903 3 0.985 4.0E-05 0.0052 100

B. Connected perturbagens (Top10) Percent rank cmap name mean n enrichment p specificity non-null 1 phenoxybenzamine 0.802 4 0.971 0 0.0891 100 2 valinomycin 0.885 4 0.960 0 0.0116 100 3 erastin 0.697 4 0.944 0 0.0000 100 4 withaferin A 0.688 4 0.943 0 0.0316 100 5 pyrvinium 0.869 6 0.937 0 0.0046 100 6 resveratrol 0.617 9 0.846 0 0.0196 100 7 thioridazine 0.603 20 0.668 0 0.0548 85 8 prochlorperazine 0.543 16 0.616 0 0.0437 87 9 5224221 0.951 2 0.996 2.0.E-05 0.0112 100 10 rottlerin 0.903 3 0.985 4.0.E-05 0.0052 100

- 21 - Figure Legends

Supplemental Figure S1. Effects of biguanides on UPR signaling pathway.

Immunoblot analysis of UPR signaling pathway proteins in 2DG-stressed cells.

HT1080 cells were transfected with 100 ng of 7u FLAG-tagged ATF6 plasmid

(expressed FLAG-tagged full-length p90ATF6) and treated for various times with VST or phenformin in the presence of 10 mM 2DG. Bu, buformin; Met, metformin; Phen, phenformin. For better detection of the p50ATF6/active form, 10 PM MG132 was included for 6 hours before cell collection and during exposure of cells to 2DG.

E-actin is used as the loading control.

Supplemental Figure S2. Quantitative real-time PCR of the Glucose Deprivation signature genes. Quantitative real-time PCR analysis of 16 genes in the Glucose

Deprivation signature. HeLa cells were exposed to stress conditions (5 mM 2DG, 1

Pg/mL of TM or glucose-free medium (GF)) for 15 hours. VST (1 PM) or biguanide

(Bu, 50 PM Buformin; Met, 1.5 mM Metformin; Phen, 15 PM Phenformin) was treated under glucose-free conditions. Relative expression levels were calculated by setting each of the normal expression levels from non-drug–treated cells as 1. Results shown are the means r SD of quadruplicate determinations.

Supplemental Figure S3. Overview of the Glucose Deprivation signature. The 246 probe sets (horizontal axis) sorted by cluster analysis displayed with 42 samples

(vertical axis). The cells were cultured for 15–18 hours under ER stress conditions

(red), under ER stress conditions with a UPR modulator (blue) and under normal conditions with a UPR modulator (green). The log ratio for each gene was calculated by setting the expression level in appropriate control sample as 0 (Log2 1). GF,

- 22 - glucose-free; 2DG, 2-deoxyglucose; TM, tunicamycin; Bu, buformin; Met, metformin;

Phen, phenformin. Details of the experimental conditions are provided as

Supplemental Table S1.

Supplemental Figure S4. Pattern-matching analysis of the VST/biguanide signature using the Connectivity Map database. A, The VST/biguanide signature including 25 probe sets (horizontal axis) sorted by cluster analysis displayed with 5 samples (vertical axis). The log ratio for each gene was calculated by setting the expression level in appropriate control sample as 0 (Log2 1). Phen, phenformin. B, Ranking of pyruvinium pamoate instances with the VST/biguanide signature. The permutated result from this query is provided in Supplemental Table S7.

- 23 - Supplemental Figure S1

10mM 2DG 10mM 2DG 10mM 2DG + 100PM Phen + 3PM VST

0 3 6 9 12 0 3 6 9 12 0 3 6 9 12 [hours] GRP94 GRP78

eIF2D

p-eIF2D

ATF4

Full-length p90ATF6 p50ATF6/ Active form E-actin

HT1080, pcFLAG-ATF6

- 24 - Supplemental Figure S2 (1 of 2)

ARMET CALR CDK2AP2

18 10 8 16 8 14 6 12 6 10 4 8 4 6 2 4 2 2 Relative Gene Gene Expression Relative Relative Gene Expression Relative Gene Gene Expression Relative 0 0 0 t T t n u t t t T t n u on GF G TM S e e B n GF G M ST e en Bu on GF G TM S e e B C 2D V +M Ph + Co 2D T V +M h + C 2D V +M Ph + + + GF + +P GF + + GF GF GF GF GF GF GF GF GF GF

DNAJB11 DNAJB9 DNAJC3

6 12 8

10 6 4 8

6 4

2 4 2 2 Relative Gene Gene Expression Relative Gene Expression Relative Relative Gene Gene Expression Relative 0 0 0 nt G M T et n u t T t n u nt G M T et n u o GF D T S M he +B on GF DG TM S e e B o GF D T S M he +B C 2 +V + P C 2 +V +M Ph + C 2 +V + P GF + GF + GF GF + GF GF GF GF GF GF GF GF

HSPA5/GRP78 HYOU1 NUCB2

25 14 12

12 10 20 10 8 15 8 6 6 10 4 4 5 2 2 Relative Gene Gene Expression Relative Relative Gene Expression Relative 0 Gene Expression Relative 0 0 nt G M T et n u t T t n u nt G M T et n u o GF D T S M he B on GF DG TM S e e B o GF D T S M he +B C 2 +V + P + C 2 +V +M Ph + C 2 +V + P + GF + GF GF + GF GF GF GF GF GF GF GF GF

P4HB PDIA4 SDF2L1

5 16 18 14 16 4 14 12 12 3 10 10 8 8 2 6 6 4 4 1 2 2 Relative Gene Gene Expression Relative Relative Gene Expression Relative Relative Gene Gene Expression Relative 0 0 0 nt G M T et n u t T t n u nt G M T et n u o GF D T S M he +B on GF G TM S e e B o GF D T S M he +B C 2 +V + P C 2D V +M Ph + C 2 +V + P GF + GF + + GF GF + GF GF GF GF GF GF GF GF

- 25 - Supplemental Figure S2 (2 of 2)

SEL1L TMED7 HSP90B1/GRP94

10 4 10

8 8 3

6 6 2 4 4

1 2 2 Relative Gene Gene Expression Relative Relative Gene Expression Relative 0 0 Gene Expression Relative 0 t t t T t n u t T t n u on GF G M ST e en Bu on GF G TM S e e B on GF G TM S e e B C 2D T V +M h + C 2D V +M Ph + C 2D V +M Ph + + +P GF + + GF + + GF GF GF GF GF GF GF GF GF GF

UBE2J1 ACTB

4 2

3 1.5

2 1

1 0.5 Relative Gene Expression Relative 0 Gene Expression Relative 0 t t n u t t on GF G TM ST e e B n G M ST e en Bu C 2D V +M Ph + Co GF 2D T V +M h + + + GF + +P GF GF GF GF GF GF GF

- 26 - Supplemental Figure S3

HeLa_2DG+VST HeLa_GF+Met HeLa_GF+Bu HeLa_GF+Phen HeLa_GF+VST HeLa_2DG HeLa_GF-2 HeLa_GF HeLa_TM HT1080_2DG-4 HT1080_2DG HT1080_TM HT1080_TM+VST HT1080_GF MKN74_GF HT-29_GF-2 HT29_GF HT1080_2DG+Met HT1080_2DG+Bu HT1080_2DG+Phen HT1080_2DG+VST HT1080_GF+VST HT1080_2DG+PP HeLa_VST MKN74_GF+VST HT-29_GF+VST HT1080_Bu HT1080_Met HT1080_Phen HT1080_PP HT1080_VST HT-29_VST MKN74_VST

Ratio (Log2) Low High 0.0 1.0 -1.0 > 2.0 < -2.0

- 27 - Supplemental Figure S4

A

HeLa_VST HT1080_VST HT1080_Phen HT-29_VST MKN74_VST

Ratio (Log2) Low High 0.0 1.0 -1.0 > 2.0 < -2.0

B 1 Pyrvinium 1PM

6100

- 28 - References

1. Park HR, Tomida A, Sato S, et al. Effect on tumor cells of blocking survival response to glucose deprivation. J Natl Cancer Inst 2004; 96: 1300-10.

2. Dennis G, Jr., Sherman BT, Hosack DA, et al. DAVID: Database for

Annotation, Visualization, and Integrated Discovery. Genome Biol 2003; 4: P3.

3. Lamb J, Crawford ED, Peck D, et al. The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and disease. Science

2006; 313: 1929-35.

- 29 -