Supplemental data:

A.1 Primer sequences used for SYBR Green real time RT-PCR

Table A.1: Primer sequences used for quantitative SYBR Green real time RT-PCR for validation of putatively differentially expressed in MDA-MB-468 cells depending on HER2 expression.

Gene Accession Primer sequence (5´3´) product # HDACfor GACTTCTACCAACCCACGTGCA NM_004964 HDACrev TTCAACGCATTCCCCATGC API5for TCCAGGGTAAAACGGGTGAG NM_006595 API5rev CAACGACTTTAATCTTGTTCTCTTCTGT ANXA4for GCTCTGCTGGCTATAGTAAAGTGCA NM_001153 ANXA4rev CGAGAAACCATCACTCTGATGAGG DTX4for GCATTCAGGGACCGGAACAC XM_166213 DTX4rev CACGAGCAGCAGCTTCAGAACTT HIF1Afor GATGAAAGAATTACCGAATTGATGG NM_181054 HIF1Arev GTCCTGTGGTGACTTGTCCTTTAGT NR4A2for CGCGGAGAACTCCTAAGGAGG NM_173171 NR4A2rev AACACAAGGCATGGCTTCAGC ZNF165for CCTATGGGACTGTGATAATGAGAGTG NM_003447 ZNF165rev CCAGCTTTGGCATGGATCTACT P4HBfor TCCTCTTTAAGAAGTTTGATGAAGGC NM_000918 P4HBrev CTGTCTGCTCGGTGAACTCGAT TOB1for TGTGTTTGCAGCCTATGGAGG NM_005749 TOB1rev AACAGGCTGGAATTGCTGGTTA SPINT2for GTGCCACTGTCACAGAGAATGC NM_021102 SPINT2rev TTCTGGGAGCACTTGGGACA BHLHB2for CACGGAGACCTACCAGGGATG NM_003670 BHLHB2rev TTGTAGGTCTCCTTGCTGTCCTC BCL2L2for GATGGTGGCCTACCTGGAGA NM_004050 BCL2L2rev AGAGCTGTGAACTCCGCCCA RARRES1for ACTTCTTCAACTTCCGGTCCG NM_206963 RARRES1rev CACCTTCCTGAAGTAAAGACTCTGG SCDfor GCTTCGAAACCGCAGTCCTC NM_005063 SCDrev TAGGAGCTAGAGATATCGTCCTGCA ELF3for GCCATGAGGTACTACTACAAACGG NM_004433 ELF3rev ACTCTGGAGAACCTCTTCCTCCT CHD3for AGGAAGACCAAGACAACCAGTCAG NM_005852 CHD3rev TGACTGTCTACGCCCTTCAGGA S100A14for kind gift of Dr. C. Kerkhoff, Experimental NM_020672 S100A14rev Dermatology, University of Münster MARK3SVfor GGAATATGCAAGTGGAGGTGAAGTA NM_002376 MARK3SVrev GTCTGTCCAGCTTGACAACCCT

A.2 Antibodies

Table A.2: Sources, pre-treatment, conditions and the dilutions for TOB, pTOB, and Epidermal Growth Factor (EGF) Antibody Source Clone Pre-treatment Dilution TOB IBL 4B1 Steamer, 1:25 citrate buffer 30´, pH 6.0 pTOB IBL polyclonal Steamer, 1:5 citrate buffer 30´, pH 6.0 EGF Acris polyclonal Steamer, TRS 1:20 buffer 30´, pH 9.0

A.3 Lists of products found to be differentially expressed in HER2 overexpressing MDA-MB-468 cells compared to control cells

Table A.3: List of 109 genes found to be upregulated in MDA-MB-468 cells overexpressing HER2 compared to the control cells containing the negative control plasmid vector. Of the 144 sequenced clones 23 could not be unambiguously identified and 12 sequences turned out to be doublets of clones identified before.

Genes upregulated in MDA-MB-468 -HER2 cDNA Accession# Locus 1 C219-reactive peptide BC047116 1 2 TARDBP: TAR DNA binding NM_007375 1p36.22 PHTF1: putative homeodomain transcription 3 factor, a gene for a protein similar to BAI1- AL133517 1p12-13.3 associated protein 4 D1S155E: NRAS-related gene NM_007158 1p22 KHDRBS1: KH domain containing, RNA 5 binding, signal transduction associated 1 (p62, NM_006559 1p32 Sam68) 6 RPS8: ribosomal protein S8 NM_001012 1p34.1-p32

7 PUM1: pumilio homolog 1 (Drosophila) NM_014676 1p35.2 8 ADIPOR1: Adiponectin receptor 1 NM_015999.2 1p36.13-q41 9 ENO1: enolase 1, (alpha) NM_001428 1p36.3-p36.2 S100A8: S100 calcium binding protein A8 10 NM_002964 1q21 (calgranulin A) 11 S100A14: S100 calcium binding protein A14 NM_020672.1 1q21.1 12 FLJ12671 NM_030980 1q22 13 IPO9: importin 9 NM_018085.3 1q31.3 14 CSRP1: cysteine and glycine-rich protein 1 NM_004078.1 1q32 PR1: voltage-dependent calcium channel 15 NM_183240.1 2q14.1 gamma subunit-like protein 16 hypothetical protein LOC130574 NM_194317 2q23.3 IDH1: isocitrate dehydrogenase 1 (NADP+), 17 NM_005896.2 2q33.3 soluble ACTR8: ARP8 -related protein 8 homolog 18 NM_022899.3 3 (yeast) 19 FLJ10618 Hypothetical protein NM_018155 3 20 RYBP: RING1 and YY1 binding protein NM_012234.3 3p14.1 IMPDH2: IMP (inosine monophosphate) 21 NM_000884 3p21.2 dehydrogenase 2 UQCRC1: ubiquinol-cytochrome c reductase 22 NM_003365 3p21.3 core protein I 23 HSPC016 NM_015933.2 3p21.31 24 RPN1: ribophorin I NM_002950.2 3q21.3-q25.2 Table A.1, continued TM4SF1: transmembrane 4 superfamily 25 NM_014220.1 3q21-q25 member 1 GALNT7: UDP-N-acetyl-alpha-D- 26 galactosamine:polypeptide N- NM_017423.1 4q31.1 acetylgalactosaminyltransferase 7 (GalNAc-T7) 27 AC020894 5 28 AC094101 5 29 AC008450 5 CCT5: chaperonin containing TCP1, subunit 5 30 NM_012073 5p15.2 (epsilon) 31 KIBRA: KIBRA protein NM_015238.1 5p35.1 32 CAST: calpastatin (TV2) NM_173060.1 5q15-q21 33 HDAC3: histone deacetylase 3 NM_003883.2 5q31 34 HTGN29: HTGN29 protein NM_020199 5q31.1 35 CSNK1A1: casein kinase 1, alpha 1 NM_001892.3 5q32 36 DKFZP566C243 AK021433.1 6p21.1 37 RDBP: RD RNA binding protein NM_002904.4 6p21.3 WBSCR22: Williams Beuren syndrome chr 38 NM_017528 7 region 22 39 DKFZP564K0822 NM_030796.2 7p11.2 EIF4H: eukaryotic translation initiation factor 4H 40 isoform / WBSCR1: Williams-Beuren syndrome NM_022170 7q11.23 region 1 41 NADH dehydrogenase (ubiquinone) 1 alpha NM_005000 7q32

subcomplex, 5, 13kDa (NDUFA5) 42 AC023102 8 UBE2V2: ubiquitin-conjugating enzyme E2 43 NM_003350.2 8q11.1 variant 2 POLR2K: polymerase (RNA) II (DNA directed) 44 NM_005034.2 8q22.2 polypeptide K, 7.0kDa 45 TIEG: TGFB inducible early growth response NM_005655 8q22.2 LY6E: lymphocyte antigen 6 complex, locus E 46 NM_002346 8q24.3 (retinoic acid induced gene E) 47 RPS6: ribosomal protein S6 NM_001010 9p21

48 FLJ12643 AK022705 9q21.31 TLE1: transducin-like enhancer of split 1 49 NM_005077.3 9q21.32 (E(sp1) homolog, Drosophila) 50 FBXW2: F-box and WD-40 domain protein 2 NM_012164 9q34 MAN1B1: mannosidase, alpha, class 1B, 51 NM_007230 9q34 member 1 SH3GLB2: SH3-domain GRB2-like endophilin 52 NM_020145.2 9q34 B2 53 AL132656 10 GHITM: growth hormone inducible 54 NM_014394 10q23.1 transmembrane protein 55 EHF: Ets homologous factor NM_012153.3 11 56 API5: apoptosis inhibitor 5 NM_006595.2 11p12-q12 Table A.1, continued 57 AP002812 11q PORIMIN: pro-oncosis receptor inducing 58 NM_052932.1 11q22.1 membrane injury gene MGST1: microsomal glutathione S-transferase 59 NM_020300 12p12.3-p12.1 1 60 CD9: CD9 antigen (p24) NM_001769.2 12p13.3 NET-5: transmembrane 4 superfamily member 61 NM_006675.2 12p13.33 tetraspan NET-5 HNRPA1: heterogeneous nuclear 62 NM_002136.1 12q13.1 ribonucleoprotein A1 63 K-ALPHA-1: tubulin, alpha, ubiquitous NM_006082 12q13.11 MYL6: , light polypeptide 6, alkali, 64 NM_021019.2 12q13.13 smooth muscle and non-muscle CCT2: chaperonin containing TCP1, subunit 2 65 NM_006431.1 12q14.3 (beta) 66 TFDP1: transcription factor Dp-1gene AF550129 13 67 AL390754 13 68 FLJ11305 NM_018386.1 13q34 69 ING1: inhibitor of growth family, member 1 NM_005537 13q34 OXA1L: oxidase (cytochrome c) assembly 1- 70 NM_005015 14q11.2 like 71 AC026583 15 72 CCNDBP1: cyclin D-type binding-protein 1 NM_012142 15q14-q15

73 hypothetical protein FLJ10036 NM_017975 15q22.2 PSMA4: proteasome (prosome, macropain) 74 NM_002789.3 15q24.1 subunit, alpha type, 4 KIF22: kinesin family member 22 / MAZ: MYC- 75 associated zinc finger protein (purine-binding NM_007317.1/NM_002383.1 16p11.2 transcription factor) 76 LITAF: lipopolysaccharide-induced TNF factor NM_004862.1 16p13.3-p12 77 FLJ13154 NM_024598.2 16q13 CES1: carboxylesterase 1 78 NM_001266.3 16q13-q22.1 (monocyte/macrophage serine esterase 1) APPBP1: amyloid beta precursor protein 79 NM_003905 16q22 binding protein 1, 59kDa 80 NQO1: NAD(P)H dehydrogenase, quinone 1 NM_000903 16q22.1 81 UBB: ubiquitin B NM_018955.2 17p12-p11.2 EIF4A1: eukaryotic translation initiation factor 82 NM_001416 17p13 4A, isoform 1 SLC25A11: solute carrier family 25 83 (mitochondrial carrier; oxoglutarate carrier), NM_003562.3 17p13.3 member 11 ERBB2: v-erb-b2 erythroblastic leukemia viral 84 oncogene homolog 2, neuro/glioblastoma NM_004448.2 17q21.1 derived oncogene homolog (avian) 85 SP6: Sp6 transcription factor NM_199262.2 17q21.3-q22 NDUFV2: NADH dehydrogenase (ubiquinone) 86 NM_021074.1 18p11.31-p11.2 flavoprotein 2, 24kDa Table A.1, continued 87 CN2: cytosolic nonspecific dipeptidase NM_018235 18q22.3 PAFAH1B3: platelet-activating factor 88 acetylhydrolase, isoform Ib, gamma subunit NM_002573.2 19q13.1 29kDa SPINT2: serine protease inhibitor, Kunitz type, 89 NM_021102.1 19q13.1 2 (placental bikunin) 90 RPS19: ribosomal protein S19 NM_001022.3 19q13.2 ITPA: inosine triphosphatase (nucleoside 91 NM_033453.2 20p triphosphate pyrophosphatase) 92 TRIB3: tribbles homolog 3 (Drosophila) NM_021158.3 20p12.2-p13 93 PCNA: proliferating cell nuclear antigen NM_002592.2 20pter-p12 94 MANBAL: mannosidase, beta A, lysosomal-like NM_022077.3 20q11.23-q12 DPM1: dolichyl-phosphate 95 mannosyltransferase polypeptide 1, catalytic NM_003859.1 20q13.13 subunit 96 cDNA FLJ90800 fis AK075281 21q22.1 GART: phosphoribosylglycinamide formyl- transferase, phosphoribosylglycinamide 97 NM_000402 21q22.11 synthetase, phosphoribosylaminoimidazole synthetase 98 DSCR3: Down syndrome critical region gene 3 NM_006052.1 21q22.2 99 ZNF278: zinc finger protein 278 NM_014323 22q12.2 100 BX293995 X 101 SMS: spermine synthase NM_004595 Xp22.1

102 PRDX4: peroxiredoxin 4 NM_006406.1 Xp22.13 103 TMSB4X: thymosin, beta 4, X chromosome NM_021109.2 Xq21.3-q22 104 MORF4L2: mortality factor 4 like 2 NM_012286.1 Xq22 105 Z83819 Xq22 TCEAL5:Transcription elongation factor A(SII)- 106 NM_032926.2 Xq22.1 like5 PSMD10: proteasome (prosome, macropain) 107 NM_002814.2 Xq22.3 26S subunit, non-ATPase, 10 108 G6PD: glucose-6-phosphate dehydrogenase NM_000402 Xq28 109 cDNA FLJ25150 fis AK057879

Table A.4: List of 112 genes found to be upregulated in MDA-MB-468 cells overexpressing HER2 compared to the control cells containing the negative control plasmid vector under EGF stimulation. Of the 144 sequenced clones 19 could not be unambiguously identified and 13 sequences turned out to be doublets of clones identified before.

Genes upregulated in MDA-MB-468 -HER2 (+EGF) cDNA Accession# Locus 1 SMYD1: SET and MYND domain containing 1 NM_198274.1 1 2 AL603750 1 3 UNR: upstream of ras NM_007158 1p22 4 PRKCL2: protein kinase C-like 2 NM_006256 1p22.2 5 CNN3: calponin 3, acidic NM_001839 1p22-p21 6 F11R: F11 receptor, NM_016946.3 1q21.2-q21.3 7 XTP2: HBxAg transactivated protein 2 NM_015172.2 1q23.3 8 XTP3TPA: XTP3-transactivated protein A NM_024096.1 1q23.4 ELF3: E74-like factor 3 (ets domain transcription factor, 9 NM_004433.3 1q32.2 epithelial-specific) 10 H3F3A: H3 histone, family 3A NM_002107 1q41 11 LBR: lamin B receptor NM_002296 1q42.1 LASS6: LAG1 longevity assurance homolog 6 (S. 12 NM_203463.1 2 cerevisiae) Similar to integrin, alpha V (vitronectin receptor, alpha 13 polypeptide, antigen CD51), clone IMAGE:5294976, BC047454 2 mRNA 14 AC009228 2 15 AC019171 2 16 AC067944 2 17 ANXA4: annexin A4 / placental anticoagulant protein II NM_001153 2p13

NR4A2: nuclear receptor subfamily 4, group A, member 2 18 NM_006186 2q22-q23 (NURR1) 19 ARHE: ras homolog gene family, member E NM_005168 2q23.3 ZAK: sterile alpha motif and leucine zipper containing kinase AZK / ETR101: immediate early protein Immediate NM_016653.1 / 2q24.2 / 20 early gene product induced by TPA stimulation in A40866 19p13.13 promyelocytic leukemia cell line HL-60 and in other leukemia cell lines 21 HSPE1 heat shock 10kDa protein 1 (chaperonin 10) NM_002157.1 2q33.1 22 NOP5/NOP58: nucleolar protein NOP5/NOP58 NM_015934.3 2q33.2 23 LRRFIP1: leucine rich repeat (in FLII) interacting protein 1 NM_004735.2 2q37.3 24 AC027125 3p 25 RPL15: ribosomal protein L15 NM_002948.2 3p24.1 CHCHD4: Coiled-coil-helix-coiled-coil-helix domain 26 NM_144636.1 3p25.1 containing 4 BHLHB2: basic helix-loop-helix domain containing, class 27 NM_003670.1 3p26 B, 2 28 BBX: bobby sox homolog (Drosophila) NM_020235.2 3q13.1 29 DIRC2: disrupted in renal carcinoma 2 NM_032839.1 3q13.33 30 TM4SF1: transmembrane 4 superfamily member 1 NM_014220.1 3q21-q25 31 GMPS: guanine monphosphate synthetase NM_003875 3q24 SERP1: Stress-associated endoplasmic reticulum protein 32 AK054644 3q25.1 1 33 PFN2: profilin 2 NM_053024.1 3q25.1-q25.2 RARRES1: retinoic acid receptor responder (tazarotene 34 NM_002888 3q25.32 induced) 1 Table A.2, continued 35 WDFY3: WD repeat and FYVE domain containing 3 NM_014991.3 4q21.3 36 ADH5: alcohol dehydrogenase 5 (class III), chi polypeptid NM_000671.2 4q21-q25 HADHSC: L-3-hydroxyacyl-Coenzyme A dehydrogenase, 37 NM_005327.1 4q22-q26 short chain 38 SNAG1: Sorting nexin associated golgi protein 1 AK022059 5 39 hypothetical protein FLJ35779 NM_152408 5q13.2

40 EGR1: early growth response 1 NM_001964.2 5q31.1 DUSP1: dual specificity phosphatase 1 (MAP kinase 41 NM_004417.2 5q34 phosphatase-1) 42 RNF130: Ring finger protein 130 NM_018434.4 5q35.1 43 SGP28: specific granule protein (28 kDa) NM_006061 6p12.3 44 ZNF165: zinc finger protein 165 NM_003447 6p21.3 45 CD24:small cell lung carcinoma cluster 4 antigen NM_013230.1 6q21 46 KIAA0680 gene product NM_014721 6q24.1 47 GARS: glycyl-tRNA synthetase NM_002047.1 7p15 48 ZDHHC4: zinc finger, DHHC domain containing 4 NM_018106.2 7p22.2 49 CD36: collagen type I receptor, thrombospondin receptor NM_000072 7q11.2 50 AKAP9: A kinase (PRKA) anchor protein (yotiao) 9 NM_147171.1 7q21-q22 51 KIAA0265 NM_014997.1 7q32.3 52 LUC7L2: LUC7-like 2 (S. cerevisiae) NM_016019.1 7q34 53 AC090154 8

ATP6V1C1: ATPase, H+ transporting, lysosomal 42kDa, 54 NM_001695 8q22.3 V1 subunit C, isoform 1 14-3-3 zeta / phospholipase A2 / protein kinase C inhibitor 55 NM_003406 8q23.1 protein-1 56 SQLE: squalene epoxidase? NM_003129 8q24.1 57 LY6E: lymphocyte antigen 6 complex, locus E NM_002346.1 8q24.4 58 ZNF216: zinc finger protein 216 NM_006007 9q13-q21 59 IARS: isoleucine-tRNA synthetase NM_002161 9q21 60 (CTSL: cathepsin L) NM_001912 9q21-q22 61 RAD23B: RAD23 homolog B (S. cerevisiae) NM_002874 9q31.2 HSPA5: heat shock 70kDa protein 5 (glucose-regulated 62 NM_005347 9q33-q34.1 protein, 78kDa) 63 ASS: argininosuccinate synthetase NM_000050.3 9q34.1 64 NET1: neuroepithelial cell transforming gene 1 NM_005863 10p15 65 RPS24: ribosomal protein S24 NM_001026 10q22-q23 66 SCD: stearoyl-CoA desaturase (delta-9-desaturase) NM_005063 10q23-q24 67 EHF: Ets homologous factor NM_012153.3 11 68 API5: apoptosis inhibitor 5 NM_006595.2 11p12-q12 69 DTX4: Deltex 4 homolog (Drosophila) AB023154.1 11q12.1 70 UEV3: ubiquitin-conjugating enzyme E2-like NM_018314.2 11q15.1 71 SLC35F2: solute carrier family 35, member F2 NM_017515 11q22.3 TIMM8B: translocase of inner mitochondrial membrane 8 72 NM_012459.1 11q23.1-q23.2 homolog B (yeast) 73 APLP2: amyloid beta (A4) precursor-like protein 2 NM_001642.1 11q23-q25 74 SLC38A1: Solute carrier family 38, member 1 NM_030674.2 12 Table A.2, continued 75 KRT 18: 18 NM_000224 12q13 76 NAP1L1: nucleosome assembly protein 1-like 1 NM_139207.1 12q21.2 77 CORO1C: coronin, actin binding protein, 1C NM_014325 12q24.1 78 RPL6: ribosomal protein L6 NM_000970.2 12q24.1 ARPC3: actin related protein 2/3 complex, subunit 3, 79 NM_005719 12q24.11 21kDa WSB2 likely ortholog of mouse WD-40-repeat-containing 80 NM_018639.3 12q24.23 protein with a SOCS box 2 hypothetical protein MGC5139 / ACADS: acyl-Coenzyme NM_032661 / 12q24.31 / 81 A dehydrogenase, C-2 to C-3 short chain NM_000017 12q22-qter 82 ARHGAP5: Rho GTPase activating protein 5 AK023014.1 14 ADPRTL2: ADP-ribosyltransferase (NAD+; poly(ADP- 83 NM_005484 14q11.2-q12 ribose) polymerase)-like 2 84 BCL2L2: BCL2-like 2 NM_004050 14q11.2-q12 HIF1A: hypoxia-inducible factor 1, alpha subunit (basic 85 NM_001530.2 14q21-q24 helix-loop-helix transcription factor) 86 ACTR10: actin-related protein 10 homolog (S. cerevisiae) NM_018477.1 14q22.2 87 HSPCA: heat shock 90kDa protein 1, alpha NM_005348.2 14q32.33 88 EMP2: Epithelial membrane protein 2 NM_001424.3 16 CES1: carboxylesterase 1 (monocyte/macrophage serine 89 NM_001266.3 16q13-q22.1 esterase 1) 90 CHD3: chromodomain helicase DNA binding protein 3 NM_001005273.1 17p13.1 91 NPEPPS: aminopeptidase puromycin sensitive NM_006310.2 17q21

92 TOB1: transducer of ERBB2, 1 NM_005749 17q21 P4HB: procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), beta polypeptide (protein disulfide 93 isomerase; thyroid hormone binding protein p55) / v-erb-a NM_000918 17q25 avian erythroblastic leukemia viral oncogene homolog 2- like 94 hypothetical protein LOC201292 NM_173547 17q25.2 95 NARF: nuclear prelamin A recognition factor NM_012336.2 17q25.3 96 PPP4R1: protein phosphatase 4, regulatory subunit 1 NM_005134.1 18p11.21 GALNT1: UDP-N-acetyl-alpha-D- 97 galactosamine:polypeptide N- NM_020474.2 18q12.1 acetylgalactosaminyltransferase 1 (GalNAc-T1) 98 SUPT5H: suppressor of Ty 5 homolog (S. cerevisiae) NM_003169 19q13 99 RPS16: ribosomal protein S16 NM_001020 19q13.1 SPINT2: serine protease inhibitor, Kunitz type, 2 (placental 100 NM_021102.1 19q13.1 bikunin) ("PKC-potentiated PP1 inhibitory protein") 101 FXYD3: FXYD domain containing ion transport regulator 3 NM_005971.2 19q13.12 HMG4L2: high-mobility group (nonhistone chromosomal) 102 NG_000979 20q11.22 protein 4-like 2 pseudogene 103 GW128: GW128 protein NM_014052 20q12-q13.12 104 STAU: staufen, RNA binding protein (Drosophila) NM_017454.1 20q13.1 OR7E23P: olfactory receptor, family 7, subfamily E, 105 NG_004368 21q22.1 member 23 pseudogene EIF3S7: eukaryotic translation initiation factor 3, subunit 7 106 NM_003753 22q13.1 zeta, 66/67kDa (DNA) 107 TUBB2: tubulin, beta, 2 NM_006088.4 X 108 FLJ12649 AK022711.1 X26.3 109 MORF4L2: mortality factor 4 like 2 NM_012286 Xq22 110 MST4: Mst3 and SOK1-related kinase NM_016542.2 Xq26.2 111 cDNA FLJ25150 fis, clone CBR07695 112 CYP3A cytochrome P450, family 3, subfamily A

Table A.5: List of 36 genes found to be downregulated in MDA-MB-468 cells overexpressing HER2 compared to the control cells containing the negative control plasmid vector. Of the 48 sequenced clones 1 could not be unambiguously identified and 10 sequences turned out to be doublets of clones identified before.

Genes downregulated in MDA-MB-468 -HER2 (+EGF) cDNA Accession# Locus 1 UMP-CMPK: UMP-CMP kinase NM_016308 1 2 BCL10: B-cell CLL/lymphoma 10 NM_003921 1p22 ACADM: acyl-Coenzyme A dehydrogenase, C-4 to C-12 3 NM_000016 1p31 straight chain KHDRBS1: KH domain containing, RNA binding, signal 4 NM_006559 1p32 transduction associated 1 (p62, Sam68) 5 NSEP1: nuclease sensitive element binding protein 1 NM_004559 1p34 6 SSB: Sjogren syndrome antigen B (autoantigen La) NM_003142 2q31.1 ALS2CR9: amyotrophic lateral sclerosis 2 (juvenile) 7 NM_025252 2q33 chromosome region, candidate 9 8 LRRFIP2: leucine rich repeat (in FLII) interacting protein 2 NM_006309 3p21.33 9 LRP11: low density lipoprotein receptor-related protein 11 NM_032832 6 10 RAN: RAN, member RAS oncogene family NM_006325 6p21 11 RANBP9: RAN binding protein 9 NM_005493 6p23 12 MTCH1: mitochondrial carrier homolog 1 (PSAP, CGI-64) NM_014341 6pter-p24.1 13 EEF1A1: eukaryotic translation elongation factor 1 alpha 1 NM_001402 6q14.1 14 cDNA: FLJ23165 fis AK026818 7 15 CGI-43: CGI-43 protein NM_015622 7p22.2 16 TPD52: tumor protein D52 NM_005079 8q21 17 PLAA: phospholipase A2-activating protein NM_004253 9p21 18 SEC61B: protein translocation complex beta NM_006808 9q22.32-q31.3 19 YME1L1: YME1-like 1 (S. cerevisiae) NM_014263 10p14 20 DKFZP564G092 NM_015601 10q22.1 21 ILK: integrin-linked kinase NM_015601 11p15.5-p15.4 22 TncRNA: trophoblast-derived noncoding RNA XM_210898 11q12.3-q13.1 23 RDX: radixin NM_002906 11q23 24 HYOU1: hypoxia up-regulated 1 NM_006389 11q23.1-q23.3 25 KRT8: NM_002273 12q13 26 MARS: methionine-tRNA synthetase NM_004990 12q13.2 27 NFYB: nuclear transcription factor Y, beta NM_006166 12q22-q23 serine/threonine protein kinase Kp78 splice variant CTAK75a AF159295 / 28 14q32.2 (MARK3: MAP/microtubule affinity-regulating kinase 3) X03205 29 RPS2: ribosomal protein S2 NM_002952 16p13.3 30 FLJ10520: hypothetical protein FLJ10520 NM_018124 16q22.2 31 RPL26: ribosomal protein L26 NM_000987 17p13 32 DDX39: DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 39 NM_005804 19p13.12 33 RPS11: ribosomal protein S11 NM_001015 19q13.3 34 SNRPB: small nuclear ribonucleoprotein polypeptides B and NM_003091 20p13

B1 35 C20orf126: chromosome 20 open reading frame 126 NM_030815 20q11.21 36 ADNP: activity-dependent neuroprotector NM_015339 20q13.13-q13.2 C21orf33 Chromosome 21 open reading frame 33 37 (ES1_HUMAN ES1 PROTEIN HOMOLOG, MITOCHONDRIAL NM_004649 21q22.3 PRECURSOR (PROTEIN KNP-I) (GT335 PROTEIN)

Table A.6: List of 40 genes found to be downregulated in MDA-MB-468 cells overexpressing HER2 compared to the control cells containing the negative control plasmid vector under EGF stimulation. Of the 48 sequenced clones 3 could not be unambiguously identified and 5 sequences turned out to be doublets of clones identified before.

Genes downregulated in MDA-MB-468 -HER2 (+EGF) cDNA Accession# Locus 1 TACSTD2: tumor-associated calcium signal transducer 2 NM_002353 1p32-p31 2 hypothetical protein PRO1489 NM_018584 1p36.12 3 ENAH: enabled homolog (Drosophila) NM_018212 1q42.13 HNRPU: heterogeneous nuclear ribonucleoprotein U (scaffold 4 NM_004501 1q44 attachment factor A) Similar to integrin, alpha V (vitronectin receptor, alpha 5 BC047454 2 polypeptide, antigen CD51) 6 SUCLG1: succinate-CoA ligase, GDP-forming, alpha subunit NM_003849 2p11.2 7 RPS7: ribosomal protein S8 NM_001012 2p26 8 hypothetical protein LOC51240 NM_016467 2q32.3 9 EIF4EL3: eukaryotic translation initiation factor 4E-like 3 NM_004846 2q37.1 10 ATR: ataxia telangiectasia and Rad3 related NM_001184 3q22-q24 11 AC012504 4 PAICS: phosphoribosylaminoimidazole carboxylase, 12 phosphoribosylaminoimidazole succinocarboxamide NM_006452 4pter-q21 synthetase RPS3A:ribosomal protein S3A/v-fos transformation effector 13 NM_001006 4q31.2-q31.3 protein 1 14 HSPA9B: heat shock 70kDa protein 9B (mortalin-2) NM_004134 5q31.1 15 DDX16: DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 16 NM_003587 6p21.3 16 EEF1A1: eukaryotic translation elongation factor 1 alpha 1 NM_001402 6q14.1 17 CCNC: cyclin C NM_005190 6q21 18 LOC55862: uncharacterized hypothalamus protein HCDASE NM_018479 6q22.33 19 AC073347 7 20 cDNA: FLJ22720 fis AK026373.1 7p15 21 ACTB: actin, beta NM_001101 7p15-p12 22 AKAP9: A kinase (PRKA) anchor protein (yotiao) 9 NM_005751 7q21-q22 23 KIAA0062 protein XM_046677 8p21.2 24 PABPC1: poly(A) binding protein, cytoplasmic 1 NM_002568 8q22.2-q23 25 FZD6: frizzled homolog 6 (Drosophila) NM_003506 8q22.3-q23.1 26 14-3-3 zeta / phospholipase A2 / protein kinase C inhibitor NM_003406 8q23.1

protein-1 27 RPS6: ribosomal protein S6 NM_001010 9p21 TIAL1:TIA cytotoxic granule-associated RNA binding protein- 28 NM_003252 10q like 1 PSAP: prosaposin (variant Gaucher disease and variant 29 NM_002778 10q21-q22 metachromatic leukodystrophy) Table A.3, continued 30 hypothetical protein FLJ13081 NM_024834 10q26.13 HSPCA: heat shock 90kDa protein 1, alpha/ PRO1073 protein/ 31 11cen-q12.3 APB_HUMAN Apolipoprotein B-100 precursor (Apo B-100) 32 PRO1073 protein AF203815 11cen-q12.3 33 TncRNA: trophoblast-derived noncoding RNA XM_210898 11q12.3-q13.1 PORIMIN: pro-oncosis receptor inducing membrane injury 34 NM_052932 11q22.1 gene 35 TXNDC: thioredoxin domain containing NM_030755 14q21.3 36 MIR16: membrane interacting protein of RGS16 NM_016641 16p12-p11.2 GGA3: golgi associated, gamma adaptin ear containing, ARF 37 NM_014001 17q25.2 binding protein 3 38 SEC13L: sec13-like protein NM_031216 18p11.1 39 RPS4X: ribosomal protein S4, X-linked NM_001007 Xq13.1 40 MORF4L2: mortality factor 4 like 2 NM_012286 Xq22

A.4 Relative expression quantification of SSH hits by quantitative RT- PCR

Table A.7: Comparison of SSH results and real time RT-PCR data for methodological evaluation. The second column names the the two cell lines which were compared in the SSH experiment leading to the identification of the cDNA given in column 1. The third column indicates which one of the compared cell lines where found to display the higher expression of the respective gene product. The list is sorted in the order of the fold changes determined by real time RT-PCR. Two cDNAs were real false positives, while 8 more revealed small expression differences by real time PCR, which are within standard

deviation. Half of the quantified cDNAs (i.e. 10) are verified to have a minimum two- fold differential expression like indicated by SSH analysis. “HER2” represents MDA- HER2 cells while “NEO” represents MDA-NEO cells.

Relative expression as indicated by real-time RT-PCR data Differential expression Relative Standard as indicated cDNA Cell line expression deviation by SSH HER2+EGF ↑ HIF1A 0,58 0.27 NEO +EGF ↓ HER2+EGF ↑ ZNF165 0,73 0.19 NEO +EGF ↓ HER2+EGF ↑ BHLHB2 1,17 0.42 NEO +EGF ↓ HER2+EGF ↑ CHD3 1,19 0.38 NEO +EGF ↓ HER2 ↑ HDAC 1,22 0.26 NEO ↓ HER2 ↑ API5 1,24 0.28 NEO ↓ HER2+EGF ↑ ANXA4 1,28 0.30 NEO +EGF ↓ HER2 ↑ S100A14 1,39 0.54 NEO ↓ HER2+EGF ↑ BCL2L2 1,49 0.49 NEO +EGF ↓ HER2+EGF ↑ ELF3 1,59 0.59 NEO +EGF ↓ HER2+EGF ↑ DTX4 2,04 0.19 NEO +EGF ↓ HER2+EGF ↑ P4HB 2,26 0.11 NEO +EGF ↓ HER2+EGF ↑ SCD 2,31 0.59 NEO +EGF ↓ HER2+EGF ↑ SPINT2 2,42 0.14 NEO +EGF ↓ HER2+EGF ↑ NR4A2 2,43 0.33 NEO +EGF ↓ HER2+EGF ↑ TOB1 4,59 0.70 NEO +EGF ↓ HER2+EGF ↑ RARRESI 5,29 0.52 NEO +EGF ↓ NEO ↑ Detectable vs non- MARK3SV ↓ n/a HER2 detetable

A.5 Differentially expressed gene products by function

Table A.8: Overview of adhesion, calcium signalling and cytoskeleton structure/ reorganization related gene products found to be upregulated in MDA-MB-468-HER2 upon EGF stimulation by SSH analysis compared to control cells.

Protein Full name Designated function name EMP2 epithelial membrane protein 2 tetraspan retinoic acid receptor responder (tazarotene RARRES1 adhesion induced) 1 CD36 collagen type I receptor, thrombospondin receptor adhesion/cytoskeleton ANXA4 annexin A4/ placental anticoagulant protein 2 calcium signaling CNN3 calponin 3, acidic cytoskeleton PFN2 profilin 2 cytoskeleton KRT 18 cytoskeleton CORO1C coronin, actin binding protein, 1C cytoskeleton ARPC3 actin related protein 2/3 complex, subunit 3, 21kDa cytoskeleton ACTR10 actin-related protein 10 homolog (S. cerevisiae) cytoskeleton procollagen-proline, 2-oxoglutarate 4-dioxygenase P4HB cytoskeleton (proline 4-hydroxylase), beta polypeptide TUBB2 tubulin, beta, 2 cytoskeleton