2 Materials and Methods

2.1 Materials

2.1.1 Chemicals

↓14

All chemicals used in this work were purchased from Roche Diagnostics GmbH, Mannheim, Germany; Calbiochem, CA, USA; Sigma-Aldrich Chemie GmbH, Munich, Germany; Sigma, MS, USA; Biozym Diagnostik GmbH, Oldendorf, Germany; Merck, Darmstadt, Germany; Serva Electrophoresis GmbH, Heidelberg, Germany; R&D Systems Inc., MN, USA.

Milli-Q 18.2 MΩ · cm water was used in all procedures if required.

2.1.2 Kits

5’-RACE Kit

BD Biosciences, CA, USA

BEGM Bullet Kit

Clonetics, San Diego, CA, USA

ChemMate Detection Kit

DAKO, Glostrup, Denmark

Developer RP X-OMAT EX

Eastman Kodak Company, NY, USA

Dual-Luciferase Reporter Assay System

Promega, WI, USA

ECL Western Blotting Detection Kit

Amersham Pharmacia Biotech, Freiburg, Germany

Endofree Plasmid Maxiprep Kit

Qiagen GmbH, Hilden, Germany

ExpressHyb solution

BD Biosciences, CA, USA

FastTrack 2.0 Kit for Isolation of mRNA

Invitrogen, CA, USA

Fixer RP X-OMAT LO

Eastman Kodak Company, NY, USA

Megaprime DNA Labelling System

Amersham Pharmacia Biotech, Buckinghamshire, UK

Opti-MEM I Reduced Serum Medium

Invitrogen, CA, USA

Plasmid Mini and Maxi Kit

Qiagen GmbH, Hilden, Germany

Protease Inhibitors Cocktail

Roche Diagnostics GmbH, Mannheim, Germany

QIAquick PCR Purification Kit

Qiagen GmbH, Hilden, Germany

QIAquick Nucleotide Removal Kit

Qiagen GmbH, Hilden, Germany

Qiaquick Gel Extraction Kit

Qiagen GmbH, Hilden, Germany

Rapid Ligation Kit

Roche Diagnostics GmbH, Mannheim, Germany

SequaGel XR

National Diagnostics, GE, USA

Thermoscript RT-PCR System

Invitrogen, CA, USA

Thermo-Sequenase Fluorescent-Labeling Cycle-Sequencing Kit

Amersham Pharmacia Biotech, Buckinghamshire, UK

TRIzol

Invitrogen, CA, USA

2.1.3 Materials

↓15

Hybond-N membrane

Amersham Pharmacia Biotech, Buckinghamshire, UK

Hybond-N+ membrane

Amersham Pharmacia Biotech, Buckinghamshire, UK

Hybond-P PVDF membrane

Amersham Pharmacia Biotech, Buckinghamshire, UK

Kodak X-ray Film

NEN Life Science Products, MA, USA

Hybond Hyperfilm

Amersham Pharmacia Biotech, Buckinghamshire, UK

Human Multiple Tissue Northern (MTN) Blot

BD Biosciences, CA, USA

Cancer Profiling Array

BD Biosciences, CA, USA

Metaphases of normal human lymphocytes

Vysis, IL, USA

2.1.4 Enzymes

Ampli Taq DNA Polymerase

Perkin Elmer, MA, USA

Calf Intestinal Alkaline Phosphatase (CIAP)

Promega, Mannheim, Germany

Restriction Endonucleases: BamHI, XbaI, KpnI, SacI, PstI, XhoI, SalI,NotI, EcoRI

Promega, Mannheim, Germany

2.1.5 Antibodies

Anti-β-actin

Sigma-Aldrich, Inc., MS, USA

Anti-AKT

Cell Signalling Technology, Inc., MA, USA

Anti-ER

Novocastra, Newcastle, UK

Anti-ERBB2

DAKO, Glostrup, Denmark

Anti-c-JUN

Cell Signalling Technology, Inc., MA, USA

Anti-Ki-67

Dianova, Hamburg, Germany

Anti-p38

Cell Signalling Technology, Inc., MA, USA

Anti-Phospho-AKT (Ser473)

Cell Signalling Technology, Inc., MA, USA

Anti-Phospho-HSP27 (Ser82)

Cell Signalling Technology, Inc., MA, USA

Anti-Phospho-c-JUN (Ser63)

Cell Signalling Technology, Inc., MA, USA

Anti-Phospho-p38 (Thr180/Tyr182)

Cell Signalling Technology, Inc., MA, USA

Anti-Phospho-p44/42 MAPK (Thr202/Tyr204)

Cell Signalling Technology, Inc., MA, USA

Anti-Phospho-SAPK/JNK (Thr183/Tyr185)

Cell Signalling Technology, Inc., MA, USA

Anti-PR

DAKO, Glostrup, Denmark

Anti-S100A14 affinity-purified

A. Pietas, Charité, Berlin, Germany

Anti-SAPK/JNK

Cell Signalling Technology, Inc., MA, USA

Anti-pan-Ras

BD Biosciences, CA, USA

Anti-α-tubulin

Monosan, Sanbio b.v., Netherlands

Anti-V5

Invitrogen, CA, USA

FITC(DTAF)-Conjugated Goat Anti-Mouse IgG (H + L)

Jackson ImmunoResearch Laboratories, Baltimore, MD, USA

FITC(DTAF)-Conjugated Goat Anti-Rabbit IgG (H + L)

Jackson ImmunoResearch Laboratories, Baltimore, MD, USA

Horseradish Peroxidase-Conjugated Rabbit Anti-Mouse

DAKO, Glostrup, Denmark

Horseradish Peroxidase-Conjugated Goat Anti-Rabbit

Amersham Pharmacia Biotech, Buckinghamshire, UK

2.1.6 Mammalian Cell Lines

↓16

HBE

Normal human bronchial epithelial cells; Clonetics, San Diego, CA, USA

SAE

Normal human small-airway epithelial cells; Clonetics, San Diego, CA, USA

H378

SCLC; ATCC, Rockville, MD,USA

H82

SCLC; ATCC, Rockville, MD, USA

COLO 677

SCLC; German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany

H446

SCLC; ATCC, Rockville, MD, USA

CPC-N

SCLC; German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany

DMS-79

SCLC; ATCC, Rockville, MD, USA

H209

SCLC; ATCC, Rockville, MD, USA

DMS-114

SCLC; ATCC, Rockville, MD, USA

H187

SCLC; ATCC, Rockville, MD, USA

N417

SCLC; ATCC, Rockville, MD, USA

H526

SCLC metastatic; ATCC, Rockville, MD, USA

COLO 668

Brain metastasis of SCLC; German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany

SHP77

Large cell variant of SCLC; ATCC, Rockville, MD; German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany

COLO 699

ADC; German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany

DV-90

ADC; German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany

H322

ADC; ATCC, Rockville, MD, USA

D51

ADC; I. Petersen, Charité, Berlin, Germany

D54

ADC; I. Petersen, Charité, Berlin, Germany

D117

ADC; I. Petersen, Charité, Berlin, Germany

H125

ADC; ATCC, Rockville, MD, USA

H23

ADC; ATCC, Rockville, MD, USA

A549

ADC; German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany

H2228

ADC; ATCC, Rockville, MD, USA

H2030

ADC; ATCC, Rockville, MD, USA

H157

SCC; ATCC, Rockville, MD, USA

H2170

SCC; ATCC, Rockville, MD, USA

H226

SCC mesothelioma; ATCC, Rockville, MD, USA

A427

Lung Ca; German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany

BEN

Lung Ca, lymph node metastasis; German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany

D97

LCLC; I. Petersen, Charité, Berlin, Germany

HBE4-E6/E7

HBE immortalized with E6 and E7 genes of HPV-16; ATCC, Rockville, MD, USA

9442
(BET-1A)

HBE immortalized with SV40 early region; ATCC, Rockville, MD, USA

BEAS-2B

HBE immortalized with SV40 early region; ATCC, Rockville, MD, USA

YP44

HBE immortalized with SV40 early region; kindly provided by Dr. Cheng, Cancer Institute, Beijing, China

HMEC

Normal human mammary epithelial cells; Clonetics, San Diego, CA, USA

HMEB

HMEC immortalized with telomerase and SV40 early region; kindly provided by Dr. B. Weinberg, Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, USA

MCF-10A

Spontaneously immortalized mammary epithelial cells; kindly provided by
Dr. A. Gontarewicz, Charité, Berlin, Germany

MCF-7

Breast Ca; kindly provided by Dr. A. Gontarewicz, Charité, Berlin, Germany

SK-BR-3

Breast Ca; kindly provided by Dr. A. Gontarewicz, Charité, Berlin, Germany

MDA-MD-231

Breast Ca; kindly provided by Dr. A. Gontarewicz, Charité, Berlin, Germany

HT-29

Colon Ca; kindly provided by Dr. K. Jürchott, Charité, Berlin, Germany

SW-480

Colon Ca; kindly provided by Dr. K. Jürchott, Charité, Berlin, Germany

HCT-116

Colon Ca; kindly provided by Dr. K. Jürchott, Charité, Berlin, Germany

CaCo-2

Colon Ca; kindly provided by Dr. K. Kölble, Charité, Berlin, Germany

WiDr

Colon Ca; kindly provided by Dr. K. Kölble, Charité, Berlin, Germany

Lovo

Colon Ca; kindly provided by Dr. A. Siegert, Charité, Berlin, Germany

CX-2

Colon Ca; kindly provided by Dr. A. Siegert, Charité, Berlin, Germany

HRT-18

Colon Ca; kindly provided by Dr. A. Siegert, Charité, Berlin, Germany

HEP-2

Head and neck Ca; kindly provided by J. Möller, Charité, Berlin, Germany

D36-1/95

Head and neck Ca; I. Petersen, Charité, Berlin, Germany

D36-2/95

Head and neck Ca; I. Petersen, Charité, Berlin, Germany

D6/95

Head and neck Ca; I. Petersen, Charité, Berlin, Germany

D3/02

Head and neck Ca; I. Petersen, Charité, Berlin, Germany

D40/97

Head and neck Ca; I. Petersen, Charité, Berlin, Germany

IMR-90

Fetal lung fibroblasts; kindly provided by Dr. A. Gontarewicz, Charité,
Berlin, Germany

HAKAT

Immortalized keratinocytes; kindly provided by Dr. N. Fusenig, DKFZ, Heidelberg, Germany

HEK 293

Human embryonic kidney epithelial cells; kindly provided by
Dr. A. Gontarewicz, Charité, Berlin, Germany

COS-7

African green monkey kidney fibroblasts; kindly provided by Dr. I. Nazarenko, Charité, Berlin, Germany

RAW 264.7

Mouse macrophagoid immortalized cells; ATCC, Rockville, MD, USA

L-cells

Mouse fibroblasts; ATCC, Rockville, MD, USA

L-Wnt 3 cells

Mouse fibroblasts transfected with Wnt-3A; ATCC, Rockville, MD, USA

C57MG

Mouse mammary epithelial cells, kindly provided by Dr. R. Nusse, Stanford University School of Medicine, USA

2.1.7 E. coli Strain

One Shot TOP10F’

F’{lacIqTn10(TetR)} mcrAΔ(mrr-hsdRMS-mcrBC) Φ80lacZΔM15 deoR recA1 endA1

Invitrogen, CA, USA

2.1.8 RNA Samples from Mammalian Cell Lines

H596

Lung Ca; Cancer Profiling Array, BD Biosciences, CA, USA

DU 145

Prostate Ca; kindly provided by Dr. A. Gontarewicz, Charité, Berlin, Germany

PC-3

Prostate Ca; kindly provided by Dr. A. Gontarewicz, Charité, Berlin, Germany

Kato III

Metastasis of gastric Ca; kindly provided by Dr. A. Gontarewicz, Charité, Berlin, Germany

EJ

Bladder Ca; kindly provided by Dr. A. Gontarewicz, Charité, Berlin, Germany

HeLa

Cervical Ca; kindly provided by Dr. A. Gontarewicz, Charité, Berlin, Germany

Daudi

Burkitt’s lymphoma; Cancer Profiling Array, BD Biosciences, CA, USA

K562

Chronic myelogenous leukemia; Cancer Profiling Array, BD Biosciences, CA, USA

HL-60

Promyeolocytic leukemia; Cancer Profiling Array, BD Biosciences, CA, USA

MOLT-4

Lymphoblastic leukemia; Cancer Profiling Array, BD Biosciences, CA, USA

Raji

Burkitt’s lymphoma; Cancer Profiling Array, BD Biosciences, CA, USA

G361

Melanoma; Cancer Profiling Array, BD Biosciences, CA, USA

SKOV-3

Ovarian Ca; kindly provided by Dr. A. Gontarewicz, Charité, Berlin, Germany

OVCAR-3

Ovarian Ca; kindly provided by Dr. A. Gontarewicz, Charité, Berlin, Germany

HT 1080

Fibrosarcoma; kindly provided by Dr. A. Gontarewicz, Charité, Berlin, Germany

ROSE 199

Rat ovarian surface epithelial cells; kindly provided by Dr. A. Gontarewicz, Charité, Berlin, Germany

ROSE A2/1

Rat ovarian surface epithelial cells transfected with KRas; kindly provided by
Dr. A. Gontarewicz, Charité, Berlin, Germany

ROSE A2/5

Rat ovarian surface epithelial cells transfected with KRas; kindly provided by
Dr. A. Gontarewicz, Charité, Berlin, Germany

208F

Immortalized rat fibroblasts; kindly provided by Dr. A. Gontarewicz, Charité, Berlin, Germany

FE-8

HRAS-transformed derivative of 208F; kindly provided by Dr. A. Gontarewicz, Charité, Berlin, Germany

IR-4

208F cells transfected with IPTG-inducible Ras; kindly provided by
Dr. A. Gontarewicz, Charité, Berlin, Germany

2.1.9 Tissue Specimens

↓17

Tumour specimens used in this study include xenografts of the following lung cancer cell lines transplanted subcutaneously into immunodefficient mice: CPC-N, H526, H446, H82, H209, N417 (SCLC); Colo 668 (brain metastasis of SCLC); D54 (adenocarcinoma), and D97 (LCLC). They were obtained from experiments conducted by Prof. Dr. I. Petersen on tumorigenicity of various human cultured lung cancer cell lines in nude mice (unpublished results). These specimens were shock frozen in liquid nitrogen and stored at -80°C until RNA extraction.

Normal human colon tissue and tumour tissue specimens used in the immunohistochemical analysis were obtained from surgical resections at the Department of Surgery of the Charité Hospital at the Humboldt University Berlin. Operation specimens were transferred to the Institute of Pathology within 1 hour after surgical removal. Generally, no adjuvant radiotherapy or chemotherapy was applied before surgery. These specimens were shock frozen in liquid nitrogen and stored at -80°C until RNA extraction.

2.1.10 Plasmids and Expression Constructs

S100A14-V5

The expression construct was generated by PCR-amplification of the ORF of the S100A14 cDNA using H1043for-tr and H1043rev-tr primers. The amplified fragment of 316-bp was ligated into the BamHI and XbaI sites of the pcDNA3.1/V5 vector.

pcDNA3.1/V5

Invitrogen, CA, USA

D1-4 human S100A14 genomic construct

The human S100A14 genomic construct was generated by cloning of the 3173-bp positive fragment (hybridizing with S100A14 coding region, intron 2, and 3 in Southern blot analysis) produced by BamHI digestion of the PAC clone D11609 into pBluescript II KS(+) phagemid vector.

pBluescript II KS(+) phagemid vector

Stratagene, La Jolla, Canada

H1043

The DKFZp404H1043 clone containing the human full-length S100A14 cloned in pSPORT1 vector, was provided by RZPD, Heidelberg, Germany.

H1043(-)0.5/10

The human full-length S100A14 expression construct was generated by subcloning from the H1043 clone by restriction digestion with NotI and XbaI.

pGL3-Basic

The firefly luciferase reporter vector lacking eukaryotic promoter and enhancer elements upstream of the firefly luciferease gene.

pRL-TK

The Renilla luciferase reporter vector containing the herpes simplex virus thymidine kinase promoter region upstream of Renilla luciferase gene.

p500-luc

The p500-luc luciferase reporter vector was generated by restriction digestion with KpnI (multiple cloning site) and SacI (endogenous restriction site; position 496 bp in D1-4) of the S100A14 genomic construct D1-4. The fragment of 495-bp was ligated into the compatible restriction sites of the pGL3-Basic firefly luciferase reporter vector.

p300-luc

The p300-luc luciferase reporter vector was generated by restriction digestion with PstI (endogenous restriction site; position 192 bp in D14) of the p500-luc fragment. The resulting PstI-SacI(-313 bp to -12 bp) restriction fragment was amplified by PCR using the oligonucleotides Prom2b-for and Prom2b-rev. The amplified fragment of 300-bp was ligated into KpnI and SacIsites of the pGL3-Basic firefly luciferase reporter vector.

p250-luc

The p250-luc luciferase reporter vector was generated by restriction digestion with PstI (endogenous restriction site; position 192 bp in D14) of the p500-luc fragment. The resulting PstI (the multiple cloning site)- PstI (-573 bp to -313 bp) restriction fragment was amplified by PCR using the oligonucleotides Prom1b-for and Prom1b-rev. The amplified fragment of 192-bp was ligated into KpnI and SacI sites of the pGL3-Basic firefly luciferase reporter vector.

p2A-luc

The p2A-luc deletion construct was created by PCR-amplification of the fragment (-313 bp to -269 bp) of the p300-luc, using prom2A-for and prom2A-rev primers. The amplified fragment of 51-bp was ligated into the KpnI and SacI sites of the pGL3-Basic firefly luciferase reporter vector.

p2B-luc

The p2B-luc deletion construct was created by PCR-amplification of the fragment (-313 bp to -206 bp) of the p300-luc, using prom2A-for and prom2B-rev primers. The amplified fragment of 107-bp was ligated into the KpnI and SacI sites of the pGL3-Basic firefly luciferase reporter vector.

p2C-luc

The p2C-luc deletion construct was created by PCR-amplification of the fragment of the p300-luc (-208 bp to -12 bp) using prom2C-for and prom2C-rev primers. The amplified fragment of 196-bp was ligated into the KpnI and SacI sites of the pGL3-Basic firefly luciferase reporter vector

EGFP (enhanced green fluorescent protein)

BD Biosciences, San Diego, TX, USA

p65/Flag

The p65 expression vector containing p65 full-length cDNA fused with Flag epitope, was a generous gift from Dr. C. Scheidereit, MDC, Berlin, Germany.

pcDNA3/Flag

The empty vector pcDNA3/Flag was a generous gift from
Dr. C. Scheidereit (MDC, Berlin, Germany).

pCR2.1 TA Cloning Vector

Invitrogen, Groningen, Netherlands

m-S100A14

The IMAGp998M1311375Q3 clone containing the mouse full-length S100A14 cDNA cloned in pCMV-SPORT6 phagemid vector, was provided by RZPD (Berlin, Germany).

2.1.11 Oligonucleotides

↓18

IRD-labelled oligonucletides were purchased from MWG-Biotech, Ebersberg, Germany. Non-labelled oligonucleotides were purchased from BioTeZ GmbH, Berlin-Buch, Germany.

Oligonucl.

Labelling

Sequence

Prom1b-for

5’– TTA GGG GTA CCC CCC AGG CAG GCT TGA GTG – 3’

Prom1b-rev

5’– TTA TAT CGA GCT CGT GCA GGG CAG GGA AGG – 3’

Prom2b-for

5’– TAG GGG TAC CCC TGC AGT TCG CCA GGG C – 3’

Prom2b-rev

5’– GCG ACC ACG AGC TCA GCT CTT ATA CCT G – 3’

18S-for

5’– GGG GAG GTA GTG ACG AA – 3’

18S-rev

5’– ACA AAG GGC AGG GAC TT – 3’

Prom2A-for

5’– TTA GGG GTA CCC CTG CAG TTC GCC AGG GCC – 3’

Prom2A-rev

5’– TTA TAT CGA GCT CGA CTT CGA GAC CTC ATG GG – 3’

Prom2B-rev

5’– TTA TAT CGA GCT CGG ATC AGC ATG CAG AGT CAC – 3’

Prom2C-for

5’– TTA GGG GTA CCC CTG CTG ATC GGA GGC CAG – 3’

Prom2C-rev

5’– TTA TAT CGA GCT CGA GCT CTT ATA CCT GGG GG – 3’

GAPDH-for

5’– GAA CGG GAA GCT TGT CAT CA – 3’

GAPDH-rev

5’– GTA GCC AAA TTC GTT GTC ATA C – 3’

mA14-for

5’– ATG GGA CAG TGT CGG TCA – 3’

mA14-rev

5’– TCA GCT CCG AGT AAC AGG – 3’

pGL3basic-f

IRD800

5’– CTA GCA AAA TAG GCT GTC CC – 3’

pGL3basic-r

IRD800

5’– CTT TAT GTT TTT GGC GTC TTC CA – 3’

exon1 for

5’– CTC AGC GGC TGC CAA CAG – 3’

exon2 for

5’– CAG TGT CGG TCA GCC AAC – 3’

exon3 for

5’– GAA CTT TCA CCA GTA CTC CG – 3’

D1-4prom-for

5’– CAT GAG GTC TCG AAG TCC – 3’

D1-4prom-rev

5’– CAG CTC ACC TGA GCT CAG – 3’

H1043for-2

5’– TCA TGC CGA GCA ACT GTG G – 3’

H1043rev-2

5’– GCC TCT CCA GCT TCA CAC T – 3’

H1043for-3

5’– GCT GCC AAC AGA TCA TGA – 3’

H1043rev-3

5’– TTG GCT GAC CGA CAC TGT – 3’

H1043 f

5’– CAA CAG AAC TCT CAC CAA AG – 3’

H1043 r

5’– TCC AGA GGG AGT TCT CAG T – 3’

H1043 i1 for

5’– GGC TGC CAA GTA AGG AAA C – 3’

H1043 i1 rev

5’– TCA TGA TCT GCT TAG AGG AG – 3’

H1043 i2 for

5’– AAC GCA GAG GTG GGC TCA T – 3’

H1043 i2 rev

5’– CTG AGC ATC CTG AGG GCA G – 3’

H1043 i3 for

5’– TCT CAT GCC GGT ATG GAC – 3’

H1043 i3 rev

5’– CCA CAG TTG CTC TGA GGG – 3’

H1043for-tr

5’– CGG GAT CCC GCA CCA TGG GAC AGT GTC GG – 3’

H1043rev-tr

5’– GCT CTA GAG CGT GCC CCC GGA CAG GCC T – 3’

GPS1

5’– AGG CCT CTC CAG CTT CAC ACT CTT G – 3’

NGPS1

5’– CAA CAG AAC TCT CAC CAA AG – 3’

NGPS2

5’– GCC TCT CCA GCT TCA CAC T – 3’

D1-4 T7-1

IRD800

5’– AGG CTG CTG CAA TAG CAG – 3’

D1-4 T7-2

IRD800

5’– TCT AAG CAG ATC ATG AGC – 3’

D1-4 T7-3

IRD800

5’– CAT CTC ATG CCG GTA TGG – 3’

H1043 M13

IRD800

5’– TGA GGT CAG ATC TCA GAA C – 3’

H1043 T7

IRD800

5’– CCT ACT TAT AAA CTC CCT A – 3’

pcDNA3.1R

IRD800

5’– CCT CGA CTG TGC CTT CTA – 3’

pcDNA3.1R-2

IRD800

5’– TAG AAG GCA CAG TCG AGG – 3’

M13

IRD800

5’– GTA AAA CGA CGG CCA G – 3’

M13R

IRD800

5’– CAG GAA ACA GCT ATG AC – 3’

T7

IRD800

5’– TAA TAC GAC TCA CTA TAG GG – 3’

2.2 Methods

2.2.1 Bacterial Culture

2.2.1.1 Routine Culturing and Storage Conditions

E.coli cultures were routinely grown at 37°C on Luria Bertani (LB) agar or in the LB broth, containing Ampicillin (50 μg/ml) or Kanamycin (25 μg/ml).

↓19

For long-term storage, a fresh overnight culture was prepared as follows: bacteria were streaked onto an agar plate containing the selective antibiotic, and incubated at 37°C overnight. Individual bacterial colonies were picked into Greiner tubes (Greiner Bio-One, Germany) containing 3 ml LB broth, and incubated for 1416 hours at 37°C with shaking at 160 rpm. Then 800 μl of 2 x bacteria storage medium and 800 μl of bacterial culture were added into the storage tube. The vials were immediately transferred to -70°C and stored until use. To recover a bacteria culture, a small amount of the frozen stock was inoculated into 3 ml LB broth, containing the appropriate antibiotic, and incubated overnight at 37°C with shaking at 160 rpm.

2 x Bacteria storage medium

8.25 g K2HPO4*3H2O

↓20

1.80 g KH2PO4

0.45 g Na-citrate*2H2O

0.09 g MgSO4*7H2O

↓21

5.90 g (NH4)2SO4

35.75 ml 87% Glycerol

sterile H2O was added to a final volume of 500 ml and the medium was filter-sterilised under sterile conditions.

↓22

1 x LB broth

1% Bacto tryptone

0.5 % Bacto yeast extract

↓23

1% NaCl

1 x LB agar

LB broth with 1.5% Bacto agar

↓24

To prepare agar plates, the LB agar was cooled to ~50°C, appropriate antibiotics were added and 15 ml of LB agar per 100-mm plate were poured. For βgalactosidase assay, LB agar plates were prepared with 80 μg/ml X-gal (5bromo-4-chloro-3-indol-β-D-galactopyranisode) and 20 mM IPTG (isopropyl-I-thio-β-D-galactopyranisode). IPTG was prepared in sterile water, and X-gal in dimethylformamide (DMF).

2.2.1.2 Transformation

Chemically competent TOP10F’ E.coli were thawed on ice and mixed gently. The cells were transformed with an aliquot of 2 µl of the ligation reaction and incubated for 30 min on ice. Next, the cells were incubated for 30 sec in the 42°C water bath and placed on ice. 250 µl of pre-warmed SOC medium (Invitrogen) were added, the vials were placed horizontally and shaken at 220 rpm and 37°C for 1 hour in a rotary shaker incubator. Finally, 50 and 200 µl of each transformed culture were spread on separate LB agar plates containing 50 µg/ml ampicilin and incubated overnight at 37°C.

Individual transformants were selected for plasmid miniprep DNA isolation by Plasmid Mini Kit and analysed by restriction endonuclease digestion and sequencing. The plates were sealed with parafilm and stored at 4°C for up to four weeks.

2.2.2 Culturing of Mammalian Cells

2.2.2.1 Routine Culturing

↓25

Mammalian cell lines were maintained at 37°C and 95% humidity in the presence of 5% CO2. Cells were typically grown in T-75 flasks and 100-mm dishes (BD Biosciences).

The cell lines used in this study were cultivated in the following media:

Cell line

Medium

HBE

BEGM

SAE

SAGM

HBE-E6/E7

BEGM

9442 (BET-1A)

BEGM

BEAS-2B

BEGM

YP44

BEGM

H378

RPMI + 10% FCS

H82

RPMI + 10% FCS

COLO 677

RPMI + 10% FCS

H446

RPMI + 10% FCS

CPC-N

McCoys + 10% FCS

DMS-79

RPMI + 10% FCS

H209

RPMI + 10% FCS

DMS-114

Waymouth's + 10% FCS

H187

RPMI + 10% FCS

N417

RPMI + 10% FCS

H526

RPMI + 10% FCS

COLO 668

RPMI + 10% FCS

SHP77

RPMI + 10% FCS

COLO 699

RPMI + 10% FCS

DV-90

RPMI + 10% FCS

H322

RPMI + 10% FCS

D51

Leibovitz 15 medium (L-15) + 10% FCS + 1% glutamine

D54

L-15 + 10% FCS + 1% glutamine

D117

L-15 + 10% FCS + 1% glutamine

H125

RPMI + 10% FCS

H23

RPMI + 10% FCS

A549

F12/Ham + 10% FCS

H2228

RPMI + 10% FCS

H2030

RPMI + 10% FCS

H157

RPMI + 10% FCS

H2170

RPMI + 10% FCS

H226

RPMI + 10% FCS

A427

RPMI + 10% FCS

BEN

Dulbecco modified Eagle’s medium (DMEM) + 10% FCS + 2 mM glutamine + 4.5 g/l glucose

D97

L-15 + 10% FCS + 1% glutamine

HMEC

MEBM

HMEB

MEBM + G418 (0.4 mg/ml) + hygromycine (0.1 mg/ml) + puromycine (1 ng/ml)

MCF 10A

DMEM:F12 HAM + 5% horse serum + 10 µg/ml insulin + 5 µg/ml hydrocortisone + 20 ng/ml EGF + 100 ng/ml cholera toxin

MCF-7

DMEM + 10% FCS + 2 mM glutamine

SK-BR 3

DMEM + 10% FCS + 2 mM glutamine

MDA MD 231

DMEM + 10% FCS + 2 mM glutamine

HT-29

L-15 + 10% FCS + 1% glutamine

SW-480

L-15 + 10% FCS + 1% glutamine

HCT-116

L-15 + 10% FCS + 1% glutamine

CaCo-2

RPMI + 10% FCS

WiDr

RPMI + 10% FCS

Lovo

DMEM + 10% FCS + 2 mM glutamine + 4.5 g/l glucose

CX-2

RPMI + 10% FCS

HRT-18

RPMI + 10% FCS

HEP-2

RPMI + 10% FCS

D36-1/95

L-15 + 10% FCS + 1% glutamine

D36-2/95

L-15 + 10% FCS + 1% glutamine

D6/95

L-15 + 10% FCS + 1% glutamine

D3/02

L-15 + 10% FCS + 1% glutamine

D40/97

L-15 + 10% FCS + 1% glutamine

IMR-90

DMEM + 20% FCS + 2 mM glutamine

HAKAT

DMEM + 10% FCS + 2 mM glutamine

HEK 293

DMEM + 10% FCS + 2 mM glutamine + 4.5 g/l glucose

COS-7

DMEM + 10% FCS + 2 mM glutamine

RAW 264.7

DMEM + 10% FCS + 2 mM glutamine + 4.5 g/l glucose

L-cells

DMEM + 10% FCS + 2 mM glutamine + 4.5 g/l glucose

L-Wnt 3 cells

DMEM + 10% FCS + 2 mM glutamine + 4.5 g/l glucose + G418 (400 µg/ml)

C57MG

DMEM + 10% FCS + 2 mM glutamine + 4.5 g/l glucose + 10 µg/ml insulin

↓26

For sub-culturing of the cells, the medium was removed, and the cells were rinsed once with 1 x PBS buffer. Then, the buffer was removed and 2 ml of trypsin-EDTA solution was added per 75-cm2 culture flask. The cells were incubated for a few minutes at 37°C until they detached. Then, 8 ml of a fresh culture medium was added, aspirated, and dispensed into new culture flasks. Fresh medium was added every 2 to 3 days.

Trypsin-EDTA 10 x stock solution (Biochrom AG) contained 0.5% Trypsin and 0.2% EDTA. For use in cell culture, the solution was diluted 1:10 with PBS, filter-sterilized, and frozen at -20°C in 80 ml aliquots. Before use, an aliquot was thawed in a water bath at 37°C and used as described above.

10 x PBS

↓27

2 g/l KCl

2 g/l KH2PO4

21.6 g/l Na2HPO4*7 H2O

↓28

pH was adjusted to 7.4

2.2.2.2 Freezing and Thawing Procedure

Following tripsinization, the cells were transferred into a 15 ml Falcon tube and centrifuged at 1 000 x g for 5 min at room temperature. The cell pellet was resuspended in 1 ml of storage medium, transferred into a cryo-tube and placed in a cryo-container with isopropanol. The cells were kept at -70°C for 24 hours to allow them to freeze slowly. After this time, they were stored in liquid nitrogen. Storage medium consisted of the respective cell culture medium supplemented with 5% DMSO.

Thawing of cells was performed quickly in a 37°C water bath for ~2 min. Then, the cells were transferred immediately into a culture flask containing pre-warmed culture medium. The next day, the medium was replaced with the fresh one to remove traces of DMSO.

2.2.2.3 Cell Treatments

↓29

For the treatment with EGF, TGF-α, and TNF-α, 9442 (BET-1A) cells were grown to ~70% confluence in regular medium (BEBM) supplemented with growth factors (BEGM) for 3 days. After this time, subconfluent cultures were treated with 50 ng/ml of human recombinant EGF, 20 ng/ml of human recombinant TGF-α, and 25 ng/ml of human recombinant TNF-α without medium change, cultured for the indicated times, and harvested. The growth and treatment of cells as well as the RNA and protein extraction were performed at least 3 independent times.

For the stimulation with medium, BEBM medium was supplemented with a double set of growth factors (BEGM; we refer to this as “stimulation medium”). After 3 days of incubation in regular culture medium, the medium was replaced with the “stimulation medium” and cells were cultured for the indicated times and harvested.

To determine the kinetics of MAPK pathways stimulation, 9442 cells were grown in regular medium for 3 days and then stimulated for various periods of time (5, 15, 30, 60, 90, 120 min) with “stimulation medium”.

↓30

In order to determine the effective drug concentration, 9442 cells were grown in regular medium for 3 days and then preincubated for 1 hour with 10 µM AG1478, 20 µM U0126, 40 µM SB203580, 40 µM SP600125, 40 µM LY294002, respectively, without medium change. Next, the cells were stimulated with “stimulation medium” for the optimal time course determined from the stimulation kinetics studies. The drug concentrations were chosen based on maximal suppressive efficacy and minimal cell toxicity. Stock solutions of 15.8 mM AG1478, 10 mM U0126, 25 mM SB203580, and 22.7 mM SP600125 were prepared in DMSO, and 50 mM LY294002 in 100% ethanol. As a control, cells were treated with the vehicle, DMSO or ethanol, respectively.

For inhibition studies, 9442 cells were incubated for 12 hours (RNA analysis) and 24 hours (protein analysis) with EGF or TGF-α without medium change, in the presence or absence of the inhibitors, which were added 1 hour prior to treatment with the growth factors.

To inhibit protein synthesis, cells were pretreated for 1 hour with cycloheximide (210 µg/ml) before adding EGF. Stock solution of 100 mg/ml cycloheximide was prepared in DMSO.

↓31

Protein kinase C (PKC) was induced by treatment with phorbol 12-myristate 13acetate (PMA; 10 nM and 100 nM) for the indicated times. Inhibition of PKC was carried out by preincubating cells for 1 hour with a broad PKC inhibitor bisindolylmaleimide I (5 µM) before stimulation with PMA or EGF. Stock solutions of 2 mg/ml PMA and 3 mM bisindolylmaleimide I were prepared in 100% ethanol and DMSO, respectively. Control plates were treated with the vehicle.

For the treatment with thapsigargin, cells were preincubated with the stimulation buffer containing 1 mM CaCl2 for 10 min (Davey et al., 2001). Then, 0.5 µM thapsigargin was added and the cells were incubated for 15 and 30 min at 37°C, fixed and immunostained for the endogenous S100A14 protein. Stock solution of 1.5 mM thapsigargin was prepared in DMSO. Control incubations were performed with DMSO as the vehicle. For Zn2+ treatment, cells were incubated with 40 µM of ZnCl2 for 15 and 30 min at 37°C.

Stimulation buffer

↓32

140 mM NaCl

5 mM KCl

1 mM MgCl2

↓33

10 mM Glucose

10 mM HEPES

1 mM CaCl2

↓34

H2O was added and the solution was filter-sterilised.

Lung tumour cell lines (H525, COLO 668, COLO 667, H157, SHP-77, D117, H23, N417) were treated with 20 μM 5-aza-2’-deoxycytydine for 5 days and processed for RNA. Stock solution of 10 mM 5-aza-2’-deoxycytydine was prepared in 1 x PBS, pH 6.0.

Stimulation of HEK 293 and Lovo cells with EGF and TNF-α was performed 24 hours after the transfection with p2C promoter construct, following 24 hours incubation in serum-containing, or serum-free medium. Cells were incubated for 6, 12, and 24 hours in the presence or absence of EGF and TNF-α and harvested after indicated times.

2.2.2.4 Transfection of Mammalian Cells

↓35

Cationic lipid-mediated transfection was employed as a method of introducing DNA into cells.

A total of 6 x 104 cells/well of the H157, A549, and COS-7 cells were seeded in 6well plates a day before transfection. The cells were transfected with 0.8 µg (H157), 1.9 µg (COS-7), and 3.8 µg (A549) of S100A14-V5 construct using 5.2 µl of Lipofectin reagent (Invitrogen, MD, USA), 6 µl, and 12 µl of LipofectAMINE reagent (Invitrogen, MD, USA), respectively. Transfection was performed according to the following protocol: for each transfection, DNA and Lipofectin/LipofectAMINE were diluted into separate aliquots (200 µl) of Opti-MEM I Reduced Serum Medium (Invitrogen). Diluted Lipofectin Reagent was allowed to stand at room temperature for 30 min. The two solutions were then combined, mixed gently and incubated at room temperature for 15 min (Lipofectin) or 45 min (LipofectAMINE) for lipid-DNA complexes formation. The cells were washed once with Opti-MEM, then 800 µl of Opti-MEM was added per well and the cells were overlaid with lipid-DNA complexes. Cells were then incubated for 5 hours at 37°C in a CO2 incubator. After this time, DNA-lipid containing medium was replaced with growth medium. Cells were assayed for gene expression 48 hours after transfection.

To control the transfection efficiency, cells were transfected with an EGFP plasmid (enhanced green fluorescent protein) and viewed 48 hours after transfection using inverted microscope equipped with UV-lamp. The number of green fluorescent cells was visually estimated. Mock-transfected cells were used as negative controls.

2.2.3 Preparation, Enzymatic Manipulation and Analysis of DNA

2.2.3.1 Mini-Preparation of Plasmid DNA

↓36

Mini-preparation of plasmid DNA was performed with Qiagen Plasmid Miniprep Kit according to the manufacturer’s recommendations. For isolation of ~15 μg of plasmid DNA, 3 ml overnight culture of E.coli in LB medium was prepared. DNA was eluted in 15 μl of the elution buffer (EB; 10 mM Tris-HCl, pH 8.5) and stored at -20°C.

2.2.3.2 Large-Scale Preparation of Plasmid DNA

For large-scale plasmid isolation, the Qiagen Plasmid Maxiprep Kit was used. Plasmid DNA preparations for transfection experiments were performed with Qiagen Endofree Plasmid Maxiprep Kit. Plasmid DNA was isolated according to the supplier’s manual. A 150 ml (Qiagen Plasmid Maxiprep Kit) or 100 ml (Qiagen Endofree Plasmid Maxiprep Kit) E.coli overnight culture was prepared for each experiment. The pellet was resuspeded in a volume of EB buffer that the final concentration of DNA was ~1.0-1.5 μg/μl.

2.2.3.3 Measurement of DNA Concentration

Concentration of DNA was measured using a GeneQuant II RNA/DNA spectrophotometer (Pharmacia Biotech). Typical OD 260/280 ratios were ~1.7-1.8.

2.2.3.4 Digestion of DNA with Restriction Endonucleases

↓37

Digestion of DNA with restriction endonucleases was performed according to the recommendations of the manufacturer. For the digestion of 1-2 μg of DNA, 10 U of enzyme were added with an appropriate buffer in a final volume of 20 μl. Reactions were incubated for 14-16 hours.

For cloning experiments, the digested DNA was subsequently separated in agarose gel, excised from the gel, and purified with QIAquick Gel Extraction Kit. Alternatively, QIAquick PCR Purification Kit was used to purify PCR-amplified fragments with incorporated restriction sites. An additional purification step for DNA was included for cloning experiments. The DNA was precipitated with 0.5 volumes of 4 M NaAc, pH 5.2 and 2.5 volumes of cold 100% ethanol, and incubated for 1 hour at -20°C. For PCR-amplified and subsequently digested DNA fragments, Pellet Paint Co-Precipitant (Novagen) was included in the precipitation reaction. Then, the solution was centrifuged at 14 000 x g and 4°C for 30 min, the pellet was washed with 1 ml 70% ethanol, and centrifuged a second time at 14 000 x g and 4°C for 5 min. Finally, the pellet was shortly air-dried and resuspeded in 10 μl of EB buffer.

2.2.3.5 Vector Dephoshorylation

To exclude self-ligation of the vector DNA, the protruding 5’-end of the vector was dephosphorylated with calf intestinal alkaline phosphatase (CIAP). The reaction was performed according to the recommendations of the supplier. One unit of CIAP was diluted for immediate use in 1 x CIAP Reaction Buffer and incubated with the vector DNA at 37°C for 30 minutes. After that time, another aliquot of diluted CIAP was added and the incubation was continued at 37°C for additional 30 minutes. The reaction was stopped by incubation at 75°C for 15 minutes. The dephosphorylated DNA was then purified by QIAquick PCR Purification Kit followed by DNA precipitation.

2.2.3.6 DNA Ligation

↓38

Ligation of DNA fragments into plasmid vectors was performed with Rapid DNA Ligation Kit. Experiments were performed according to the protocol of the supplier. A maximum of 200 ng of total DNA (vector + insert) at the molar ratios of vector DNA to insert DNA 1:2 and 1:5 were used. The reactions were incubated for 5 min at room temperature and then placed on ice. To determine the efficiency of ligation, 5 μl of the reaction mix were run on an agarose gel and visualised with ethidium bromide.

2.2.3.7 Polymerase Chain Reaction (PCR)

Amplification of DNA was performed according to the following protocol:

Template DNA

25.0 ng – 0.5 μg

10 x PCR buffer (including 15 mM MgCl2)

2.50 μl

dNTP mix (each 10 nM)

1.00 μl

5’- and 3’- oligonucleotide primers

1.00 μl (each)

AmpliTaq DNA polymerase (1U)

0.25 μl

H2O filled up to

25.0 μl

↓39

For amplification of DNA fragments from PAC genomic clone, 5% DMSO was added to the reaction.

Typical cycling profiles were programmed in Primus 96 Plus Thermal Cycler (MWG-Biotech) as follows:

Denaturation of double-stranded DNA

95°C

30 sec

 

Annealing of oligonucleotides to the template DNA

Ta

30 sec

 

Elongation

72°C

30/60 sec

 

Extension

72°C

5 min

 

Chilling

4°C

up to 1 h

 

↓40

Annealing temperatures were calculated using the formula: Ta = Tm - 2°C, where Tm is the melting temperature of the primers used for amplification. Tm of each oligonucleotide primer was calculated as follows: Tm = 4 x (G + C) + 2 x (A + T), where G – guanine, C – cytosine, T – thymine, A – adenine.

2.2.3.8 Purification of PCR-Amplified Fragments of DNA

Purification of PCR-amplified DNA fragments was performed with QIAquick PCR Purification Kit according to the supplier’s protocol. Purified PCR fragments were controlled by agarose gel electrophoresis.

2.2.3.9 Sequencing

Sequencing analysis was performed on a LI-COR automated DNA sequencer (MWG-Biotech) using fluorescent primers labelled with the tricarbocyanine dye IRD800 at their 5’-end.

↓41

Gel components were pre-mixed the following way:

30 ml Sequagel XR (Biozym)

7.5 ml Sequagel-buffer (Biozym)

↓42

150 µl 10% Ammonium persulfate (APS)

15 µl TEMED

and polymerised for 1 hour.

↓43

Cycle sequencing reactions were done using the Thermo-Sequenase Fluorescent-Labeling Cycle-Sequencing Kit according to a protocol of the supplier.

The reactions were pre-mixed the following way:

4 µl H2O

↓44

4 µl plasmid DNA (100-300 ng/µl)

4 µl IRD800-labelled primer (2 pmol/µl)

to a total reaction volume of 12 µl.

↓45

The pre-mix was distributed in four tubes, 3 µl in each, and 1.3 µl of Thermo-Sequenase DNA Polymerase and Termination Mix A/C/G/T was added to each tube.

The following parameters were used for a cycle-sequencing:

30 sec

95°C

 

15 sec

Ta

30 cycles

1 min

70°C

 

↓46

Each reaction was stopped with 4 µl of a Stop/Loading Buffer. Samples were then analysed by electrophoresis or stored at -20°C.

The reaction tubes were heated for 5 min at 70°C to denature the samples. Then 1.3 µl/well was loaded onto sequencing gel, and run overnight. Data were analysed using a LI-COR user program.

2.2.3.10 Electrophoretic Separation of DNA

Gel electrophoretic separation of DNA was performed in 1 to 2% agarose gels run at 30 to 80V with 0.5 x TBE as a running buffer. Samples were diluted in 6 x Loading Dye (Promega) and loaded on gels containing 0.1 μg/ml ethidium bromide for visualisation.

↓47

10 x TBE

108 g Tris-base

55 g Boric acid

↓48

40 ml 0.5 M EDTA, pH 8.0

adjusted with H2O to 1 l

2.2.3.11 Elution of DNA Fragments from a Gel

For elution of DNA fragments from agarose gels, the QIAquick Gel Extraction Kit was used. DNA fragments were excised from the gel on UV light and handled as recommended by the manufacturer. DNA was eluted in 30 μl of EB buffer, purified, and concentrated by ethanol precipitation.

2.2.3.12 Southern Blot Analysis

↓49

For Southern blot analysis, DNA samples were loaded onto a 1% agarose gel and run at 50V for 4 hours or overnight. Following electrophoresis, the gel was processed for blotting by depurination, denaturation, and neutralization. The gel was rinsed in H2O between each step.

For depurination, the gel was placed in 0.25 M HCl and agitated gently for 15 min. Then, the gel was submerged in denaturation buffer (0.5 M NaOH/1.5 M NaCl) and incubated for 45 min with gentle agitation. To neutralize the gel, it has been submerged in neutralization buffer (0.5 M Tris-HCl/1.5 M NaCl) and incubated for 30 min with gentle agitation. Then, the neutralization solution was replaced with the fresh one and incubated for further 30 min.

The DNA was transferred from the gel onto the Hybond-N+ membrane by vacuum transfer using the Vacuum blotter (Model 785, BIO-RAD) for 90 min. 10 x SSC was used as a transfer buffer. The membrane was pre-wetted with H2O and then submerged for 5 min in transfer buffer. Following the transfer, the membrane was rinsed for 5 min in 2 x SSC, air-dried, and cross-linked by UV exposure at 120 000 microjoules/cm2. The membrane was wrapped in plastic foil and stored at 4°C until use.

↓50

DNA probes were labelled with [α-32P] dCTP by random oligonucleotide priming using Megaprime DNA Labeling System, according to the manufacturer’s recommendations. Typically, 25 ng of labelled probe was used for each hybridization. Unincorporated nucleotides were removed using QIAquick Nucleotide Removal Kit, according to the protocol of the supplier. Subsequently, the radioactively labelled probe was denatured at 98°C for 5 min and quickly placed on ice.

Prehybridization and hybridization were carried out in glass tubes in a commercial hybridization oven. The membrane was placed RNA-side-up in a hybridization tube, ~5 ml of pre-warmed ExpressHyb solution were added and it was prehybridized with rotation at 60°C for 30 min. Following prehybridization, the denaturated probe was added into the hybridization solution and the incubation was continued at 60°C overnight.

The next day, the blot was rinsed quickly in 2 x SSPE/0.1% SDS wash solution. Then, the washing solution was replaced with the fresh one and incubated with rotation for 1 hour at room temperature. After that time, the solution solution was replaced with 0.1 x SSPE/0.1% SDS wash solution and incubated with rotation for 1 hour at 60°C. After the final wash, the membrane was immediately covered with plastic wrap and mounted. Autoradiography was performed by exposition to Kodak X-ray film at -70°C in a cassette with two intensifying screens.

↓51

For re-probing the membrane, the probe was stripped from the membrane by incubation with shaking for 10 min in 0.5% SDS solution pre-warmed to 80-100°C. The membrane was then covered with plastic wrap and stored at 4°C until use.

20 x SSC

3 M NaCl

↓52

0.3 M Natrium citrate

20 x SSPE

3.6 M NaCl

↓53

0.2 M Natrium phosphate

0.02 M EDTA, pH 8.0

pH was adjusted to 7.7, H2O was added to a final volume of 1 liter and the solution was autoclaved.

2.2.3.13 Cancer Profiling Array Analysis

↓54

Cancer Profiling Array (BD Biosciences) consists of SMART-amplified cDNA from 241 tumour and corresponding normal tissues from individual patients, along with eight negative and positive controls (yeast total RNA; yeast tRNA; E.coli DNA; human Cot-1 DNA; poly(A); ubiquitin cDNA; human genomic DNA). Samples on the array are normalized to four different housekeeping genes: ubiquitin, 23-kDa highly basic protein, β-actin, and glutamase dehydrogenase. The array also includes the following human cancer cell lines: HeLa; Burkitt’s lymphoma, Daudi; chronic myelogenous leukemia K562; promyelocytic leukemia HL-60; melanoma G361; lung carcinoma A549; lymphoblastic leukemia MOLT-4; colorectal adenocarcinoma SW480, and Burkitt’s lymphoma, Raji.

The 369-bp PCR fragment of the human S100A14 cDNA, corresponding to the complete protein coding region, was used as a hybridization probe. The probe was prepared using H1043f and H1043r primers.

Hybridization was performed according to the manufacturer’s recommendations and as described above for Southern blot, except for the following modifications: the array membrane was prehybridized in 15 ml of prewarmed ExpressHyb solution with 1.5 mg yeast tRNA at 65°C for 2 hours. The radioactively labelled cDNA probe was mixed with 100 µg of yeast tRNA, denatured at 98°C for 10 min, and hybridized to the array at 65°C overnight. Following exposure to a phosphor screen for 14 hours and 7 hours, the array was scanned with the GS-250 Molecular Imager (BIO-RAD, Munich, Germany) at a 800-µm resolution. The results were quantified using the Molecular Analyst Software Version 1.4 by applying a grid to the image to measure the intensity of the hybridization signal of every spot. After background subtraction, the image was normalized using all spots on the membrane as reference points. The threshold values for up- and down-regulation were chosen based on careful inspection of tumour/normal intensity ratios for all the spots on the membrane (intensity ratio >2 for up-regulation and <0.5 for down-regulation).

2.2.3.14 Fluorescence in situ Hybridization (FISH) Analysis

↓55

The FISH analysis was performed by Ms. N. Deutschmann (Institute of Pathology, Berlin, Germany) as described by Lichter and Ried (Godsen, 1994). The DNA from the genomic PAC clones I22230, A12752, and D11609 was purified with Qiagen Maxi Kit and labelled by nick-translation with biotin-16-dUTP (Roche). Next, hybridization on metaphases of normal lymphocytes was performed. Images were captured with a cooled charge-coupled device camera (Photometrics, Tucson, Arizona) and processed using a custom made program (Karyomed, KaryoMedics, Germany).

2.2.3.15 5’ Rapid Amplification of cDNA Ends (5’ RACE)

The 5’-end of the S100A14 cDNA was determined with 5’ RACE Kit according to the supplier’s manual, using poly(A) RNA from normal colon tissue. The gene-specific primers for 5’ RACE included GPS1, NGPS1, and NGPS2. The PCR products were cloned into the pCR2.1 TA Cloning Vector and 24 clones were sequenced.

2.2.4 Dual-Luciferase Reporter Assay

Human embryonic epithelial kidney cells HEK 293 were plated in 6-well plates at 1.5 x 105 cells/well a day before transfection. The cells were transfected for 6 hours using a transfection mixture consisting of 200 µl of Opti-MEM, 4 µl of LipofectAMINE, 1 µg of Firefly luciferase reporter constructs driven by the S100A14 promoter fragments, and 0.1 µg of pRL-TK Renilla luciferase reporter vector as an internal control for transfection efficiency.

↓56

For co-transfection experiments, cells were transfected with a total of 1 µg DNA, consisting of a mixture of 0.5 µg of the p2C-luc plasmid and 0.5 µg of the expression vector encoding wild type p65. Where necessary, “empty” vector for p65 (pcDNA3Flag) or pGL3-Basic vector were included to maintain constant amounts of DNA.

The assays for firefly and Renilla luciferase activity were performed using the Dual-Luciferase Reporter Assay Kit (Promega), as recommended by the manufacturer. Forty-eight hours after transfection, the cells were washed once with 1 x PBS and cell lysates were prepared by passive lysis in 250 µl of freshly prepared 1 x passive lysis buffer (Promega). Firefly and Renilla luciferase activities were assayed in 20 µl of cell lysate pre-dispensed into 5-ml Sarstedt tubes, followed by sequential autoinjection of 25 µl LAR II and Stop&Glo reagents using the Lumat LB9507 luminometer fitted with automatic injection system (Berthold Technologies, Germany). The luminometer was programmed to perform a 2second per measurement delay, followed by a 10-second measurement period for each reporter assay.

Luciferase read-out was obtained from triplicate transfections and averaged. Firefly luciferase activity was normalized to the Renilla luciferase activity, and the results were expressed as relative luciferase activity.

2.2.5 Preparation and Analysis of RNA

2.2.5.1 RNA Preparation

↓57

Total RNA from cultured cells was isolated using TRIzol (Invitrogen). Cells growing on 100-mm dishes were washed once with pre-cooled 1 x PBS and lysed by adding 2 ml of TRIzol reagent and passing the cell lysate several times through a pipette. Then the lysed cells were scraped off with a sterile cell scraper (Sarstedt) and transferred to 11-ml Sarstedt tubes. The samples were then extracted with 0.4 ml of chloroform (0.2 ml chloroform per 1 ml of TRIzol reagent) by inversion mixing. Phase separation was achieved by placing the samples at room temperature for 5 min, and centrifuging at 9 000 x g and 4°C for 20 min. The aqueous layer was mixed with 1 ml of isopropanol (0.5 ml isopropanol per 1 ml of TRIzol reagent) to precipitate the RNA. Samples were incubated at room temperature for 10 min, then centrifuged at 10 000 x g and 4°C for 10 min. The RNA pellet was washed with 2 ml of 80% ethanol (1 ml ethanol per 1 ml of TRIzol reagent), and centrifuged at 7 500 x g and 4°C for 5 min. The pellet was air-dried, resuspended in DEPC-treated water, and the RNA was quantified using a GeneQuant II RNA/DNA spectrophotometer (Pharmacia Biotech). The concentration of RNA was adjusted to ~1.0-1.5 μg/μl. Typical OD 260/280 ratios were ~1.4-1.5. To assess the integrity of the RNA preparations, a test formaldehyde-agarose gel electrophoresis was performed.

Tissue samples were homogenized in 4 ml of TRIzol reagent using a power homogenizer (MICRA). The homogenized samples were incubated at room temperature for 5 min and followed the standard RNA extraction procedure as outlined above.

Poly(A) RNA was isolated using FastTrack 2.0 Kit (Invitrogen) according to the recommendations of the manufacturer.

↓58

DEPC-treated H 2 O

1 l H2O

1 ml Diethylpyrocarbonate (DEPC)

↓59

The solution was stirred on a magnetic stirrer overnight in a fume hood and autoclaved the next day.

2.2.5.2 Electrophoretic Separation of RNA

A 10-µg aliquot of total RNA was size-fractionated on 1% denaturing agarose gel in the presence of 2.2 M formaldehyde.

To denature the RNA sample, a denaturation reaction was set up:

↓60

RNA

10.0 µg

10 x MOPS

1.0 µl

Formaldehyde

3.5 µl

Formamide

10.0 µl

10 x Gel-loading buffer

2.0 µl

The RNA solution was incubated for 10 min at 55°C and then quickly chilled on ice. After loading, the gel was run in 1 x MOPS electrophoresis buffer at 50V for ~5 hours, visualised and photographed on a UV transilluminator.

Denaturing agarose gel

↓61

1 g Agarose

72.2 ml of DEPC-treated H2O

agarose was disolved by boiling in a microwave oven and cooled to ~50°C. In a chemical fume hood, 10 ml of 10 x MOPS electrophoresis buffer and 17.8 ml of formaldehyde were added and the gel was casted.

↓62

10 x MOPS

0.2 M MOPS

50 mM Sodium acetate

↓63

10 mM EDTA, pH 8.0

pH was adjusted to 7.0 with NaOH

10 x Gel-loading buffer

↓64

500 μl Formamide

2 μl EDTA, pH 8.0

5 μl Ethidium bromide

↓65

0.1% w/v Bromophenol blue

2.2.5.3 Northern Blot Analysis

Electrophoretically separated RNA was transferred from gel onto Hybond-N membrane by upward capillary transfer. Prior to the transfer, the gel was rinsed with H2O. The membrane was prepared for the transfer by immersing in 10 x SSC for 5 min and the gel was placed on a solid support in an inverted position. Then, the membrane was placed on the top of the gel, followed by 6 pieces of Whatman 3MM papers and a stack of paper towels. A glass plate was put on it and weighted down with a 500-g weight. The transfer occurred overnight in 20 x SSC buffer. The next day, the membrane was immersed for 5 min in 2 x SSC, air-dried, and cross-linked by UV irradiation. If not used immediately, the membrane was stored at 4°C.

The radioactive labelling of the probe, hybridization, washing of the blots, and stripping of radiolabelled probes were carried out as described for Southern blot analysis (Section 2.2.3.12). Following modifications to the protocol were introduced for Northern blot analysis: the hybridization temperature was 64°C and the membrane washing times were 40 min. All the washing steps were performed with solutions prepared with DEPC-treated H2O. As a hybridization probe, the PCR fragment of the human S100A14 cDNA corresponding to the complete protein coding region was used. To confirm equal RNA loading and transfer, the 180-bp fragment of the mouse 18S cDNA that cross-hybridizes with human 18S RNA was used.

↓66

Typically, the blots were exposed to a phosphor screen for different time periods and scanned with the GS-250 Molecular Imager (BIO-RAD, Munich, Germany) at a 200-µm resolution. The results were quantified using the Molecular Analyst Software Version 1.4. Alternatively, if autoradiography was performed, the autoradiographs were scanned to quantify the intensity of the radiographic bands using a scanning laser BIO-RAD Laboratories Imaging Densitometer Model GS670 and densitometry was performed.

2.2.5.4 Reverse Transcriptase-PCR (RT-PCR) Analysis

For the detection and expression analysis of the S100A14 mRNA, the THERMOSCRIPT RT-PCR System (Invitrogen) was applied. First, cDNA synthesis was performed using 3 μg of total RNA primed with gene-specific primer H1043-rev and control gene primer GAPDH-rev. The RNA was reverse-transcribed at 55°C for 1 hour according to the manufacturer’s recommendations. The reaction was terminated by heating at 85°C for 5 min. In the second step, PCR amplification was carried out using 2 μl of the cDNA synthesis reaction and primed with gene specific primers designed from open reading frame: ex1-for or ex3-for, and H1043-rev. A control PCR reaction for GAPDH was performed in parallel using the primer pair: GAPDH-for and GAPDH-rev. Amplification resulted in DNA fragments of 249-bp (ex1-for and H1043-rev), 425-bp (ex1-for and H1043rev), and 800-bp (GAPDH-for and GAPDH-rev). PCR products were analysed on a 1.2% agarose gel and viewed on UV light.

2.2.6 Analysis of Proteins

2.2.6.1 Protein Isolation from Mammalian Cells

For protein isolation, cells were washed twice with cold 1 x PBS and lysed with denaturing hypotonic lysis buffer on ice. The lysed cells were scraped off the plates, transferred into 1.5-ml Eppendorf tubes, and incubated with shaking for 10 min at 95°C. Then, the protein samples were aliquoted and stored at -20°C.

↓67

Denaturing hypotonic lysis buffer

1% SDS

10 mM Tris-HCl, pH 7.5

↓68

2 mM EDTA, pH 8.0

2.2.6.2 Subcellular Fractionation

Cells were washed twice with cold 1 x PBS and lysed in hypotonic lysis buffer (1.2 ml per 100-mm plate) for 10 min. The cell lysate was homogenized in a pre-cooled Dounce homogenizer by repeated strokes and centrifuged for 10 min at 2 700 x g to pellet the nuclei using a Sorvall SA-600 rotor. The nuclear pellet was resuspended in a volume of 50 μl of 2 x SDS sample buffer.

The postnuclear supernatant was transferred into Beckman tubes (13 x 15 mm) and centrifuged for 30 min at 60 000 x g and 4°C to pellet the membranes using a Beckman TLN 100.3 rotor. The crude membranes were resuspended in a volume of 100 μl of 2 x SDS sample buffer.

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The postmembrane supernatant was further fractionated by methanol/ chloroform precipitation to recover cytoplasmic proteins. Four ml methanol were added to the postmembrane supernatant, then 1 ml chloroform was added and mixed, then 3 ml of H2O were added and mixed. The solution was centrifuged at 15 000 x g for 15 min at 4°C. The upper phase was discarded, and 6 ml methanol were added, mixed and centrifuged again at 15 000 x g for 15 min at 4°C. The pellet was dried and resuspended in 100 μl of 2 x SDS sample buffer.

Following dissolving of the pellets in 2 x SDS sample buffer, samples were incubated with shaking for 10 min at 95°C, centrifuged for 5 min at 14 000 x g, and stored at -20°C until use.

The resulting fractions were tested for their enrichment in soluble, membrane, and nuclear fractions by Western blotting with anti-α-tubulin, anti-pan RAS, and anti-phospho-c-JUN antibodies, respectively.

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Hypotonic lysis buffer

10 mM Tris-HCl, pH 8.0

0.1 mM DTT

↓71

1 mM PMSF

1 tab/10 ml protease inhibitor Complete tabs (Roche)

2 x SDS sample buffer

↓72

120 mM Tris-HCl, pH 6.8

200 mM DTT

4% w/v SDS

↓73

10% w/v Glycerol

0.002% w/v Bromophenol blue

2.2.6.3 Determination of Protein Concentration

Protein content was determined by colorimetric amido-black method as described by Schaffner and Weissmann (1973).

↓74

A standard curve for bovine serum albumine (BSA) was created by preparing 0.5, 0.75, 1, 2, 4, 8, and 10-μg duplicate dilutions of 200 mg/ml stock solution of BSA.

Then, 1 μl of protein standards (applied in duplicates) and 1 μl of protein sample were applied to the nitrocelulose membrane (Schleicher & Schüll). The membrane was placed in 0.1% amido-black for 1 min and then washed extensively with shaking using freshly prepared destaining solution. The destaining solution was replaced several times till the membrane was completely destained. The protein spots were cut out, put into Eppendorf tubes, and 800 μl of elution solution were added. The tubes were placed in an Eppendorf shaker and shaken for 30 min.

Absorbance of standards and protein samples was measured at 630 nm in disposable 1.5 ml semi-micro cuvettes (Brand) using Biochrom 4060 software in UV-Visible Spectrophotometer (Pharmacia LKB). Protein concentration was determined by plotting absorbance of the protein samples against the standard curve using Microsoft Excel program.

↓75

Destaining solution

90 ml Methanol

2 ml Acetic acid

↓76

8 ml H2O

Elution solution

50 ml Ethanol

↓77

10 μl 0.5 M EDTA, pH 8.0

0.5 ml 5 M NaOH

49.5 ml H2O

2.2.6.4 One-Dimensional SDS Gel Electrophoresis (PAGE)

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Protein samples were separated by polyacrylamide gel electrophoresis (PAGE) using the standard Laemmli method.

Protein samples were mixed with 1 volume of 2 x SDS sample buffer and denatured for 5 min at 95°C. Equal amounts of proteins were loaded onto 12% gel and run ~4 hours at 65V in 1 x running buffer using Mini-PROTEAN 3 Cell electrophoresis tank (BIO-RAD). Protein separation was controlled by running a Prestained Broad Range Protein Marker (BIO-RAD).

Separating gel: 10 ml of 12% gel

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3.35 ml H2O

2.5 ml 1.5 M Tris-HCl, pH 8.8

4.0 ml 30% Acrylamide/Bisacrylamide

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100 μl 10% SDS

50 μl APS

10 μl TEMED

↓81

Stacking gel: 5 ml of 4% gel

3.05 ml H2O

1.25 ml 0.5 M Tris-HCl, pH 6.8

↓82

0.66 ml 30% Acrylamide/Bisacrylamide

50 μl 10% SDS

50 μl APS

↓83

10 μl TEMED

5 x running buffer

15.1 g/l Tris-base

↓84

72.0 g/l Glycine

5.0 g/l SDS

An alternative protocol was used for S100A14 gel electrophoresis. Protein samples were mixed with 1 volume of 2 x tricine sample buffer and denatured for 5 min at 95°C. Equal amounts of proteins were loaded onto 15% Tris-tricine gel and run in 1 x cathode (upper tank) and 1 x anode (lower tank) electrophoresis buffer for ~1 hour at 35V and ~5 hour at 50V.

↓85

2 x Tricine sample buffer

2.0 ml 0.5 M Tris-HCl, pH 6.8

2.4 ml Glycerol

↓86

4.2 ml 20% SDS

0.31 g DTT

0.02% w/v Coomassie blue

↓87

adjusted with H2O to 10 ml

Separating gel: 10 ml of a 15% gel

1.66 ml H2O

↓88

3.32 ml 3 M Tris-HCl, pH 8.45

1.06 ml Glycerol

3.75 ml 40% Acrylamide/Bisacrylamide

↓89

150 μl 20% SDS

50 μl APS

10 μl TEMED

↓90

Stacking gel: 5 ml of 4% gel

2.71 ml H2O

1.66 ml 3 M Tris-HCl, pH 8.45

↓91

0.5 ml 40% Acrylamide/Bisacrylamide

75 μl 20% SDS

50 μl APS

↓92

10 μl TEMED

Cathode electrophoresis buffer

12.11 g/l Tris-base

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17.92 g/l Tricine

1 g/l SDS

stored at 4°C

↓94

Anode electrophoresis buffer

24.22 g Tris-base

diluted to 1 l with H2O, adjusted to pH 8.9, and stored at 4°C

2.2.6.5 Western Blot Analysis

↓95

Following electrophoresis, the stacking gel was discarded, and the separating gel was submerged in transfer buffer for 20 min. During that time, PVDF membrane (Amersham) was activated in methanol for 11 sec, washed in H2O for 5 min, and equilibrated in transfer buffer for 10 min. On a semidry transfer unit (Peqlab Biotechnology GmbH) 6 sheets of Whatman paper pre-wetted in transfer buffer, the PVDV membrane, the gel, and 6 sheets of pre-wetted Whatman paper were assembled. The protein transfer proceeded for 45 min at 200 mA.

Transfer efficiency was controlled by Coomassie blue staining of the gel for 30 min, followed by destaining and visualisation. After the transfer, the membrane was washed briefly in TBST buffer and blocked for 1 hour in 10 ml of blocking buffer at room temperature with shaking on a rotor platform. After the blocking procedure, the membrane was washed 3 x 5 min in 20 ml TBST, and incubated overnight with the primary antibody in 5% BSA solution (5% BSA in TBST) at 4°C. The next day, the membrane was washed 3 x 10 min in 20 ml TBST, and incubated with the corresponding secondary antibody for 1 hour. The membrane was washed again 3 x 10 min in TBST, and the immunocomplexes were detected with an Enhanced Chemiluminescence Kit (ECL, Amersham), as recommended by the supplier.

For S100A14 detection, the membranes were incubated with the primary antibody for 2 hours in blocking buffer at room temperature.

↓96

For re-probing, the membranes were incubated in stripping buffer for 30 min at 70°C and washed 3 x 10 min in TBST. The membranes were subsequently blocked in blocking buffer and immunoblotted with a primary antibody as outlined above.

Primary antibodies, corresponding secondary antibodies, and their dilutions

Primary antibody

Dilution

Secondary antibody

Dilution

Anti-β-actin

1:10 000

Peroxidase-conjugated rabbit anti-mouse

1:5000

Anti-AKT

1:1000

Peroxidase-conjugated goat anti- rabbit

1:5000

Anti-c-JUN

1:1000

Peroxidase-conjugated goat anti- rabbit

1:5000

Anti-p38

1:1000

Peroxidase-conjugated goat anti- rabbit

1:5000

Anti-Phospho-AKT (Ser473)

1:1000

Peroxidase-conjugated goat anti- rabbit

1:5000

Anti-Phospho-HSP27 (Ser82)

1:1000

Peroxidase-conjugated goat anti- rabbit

1:5000

Anti-Phospho-c-JUN (Ser63)

1:1000

Peroxidase-conjugated goat anti- rabbit

1:5000

Anti-Phospho-p38 (Thr180/Tyr182)

1:1000

Peroxidase-conjugated goat anti- rabbit

1:5000

Anti-Phospho-p44/42 MAPK (Thr202/Tyr204)

1:1000

Peroxidase-conjugated goat anti- rabbit

1:5000

Anti-Phospho-SAPK/JNK (Thr183/Tyr185)

1:1000

Peroxidase-conjugated goat anti- rabbit

1:5000

Anti-S100A14 affinity-purified

1:2700

Peroxidase-conjugated goat anti- rabbit

1:5000

Anti-SAPK/JNK

1:1000

Peroxidase-conjugated goat anti- rabbit

1:5000

Anti-pan-RAS

1:800

Peroxidase-conjugated rabbit anti-mouse

1:5000

Anti-α-tubulin

1:5000

Peroxidase-conjugated rabbit anti-mouse

1:5000

Anti-V5

1:5000

Peroxidase-conjugated rabbit anti-mouse

1:5000

↓97

Transfer buffer

5.81 g Tris-base

2.93 g Glycine

↓98

1.875 ml 20% SDS

200 ml Methanol

H2O was added to 1 l

↓99

TBST

10 ml 1 M Tris-base, pH 8.0

30 ml 5 M NaCl

↓100

500 μl Tween-20

H2O was added to 1 l

Coomassie blue staining solution

↓101

50% v/v Methanol

0.05% v/v Coomassie blue R-250

10% v/v Acetic acid

↓102

40% H2O

Coomassie blue destaining solution

5% v/v Methanol

↓103

7% v/v Acetic acid

88% H2O

Blocking buffer

↓104

5% Non-fat dry milk (Difco, Becton Dickinson, MD, USA) in TBST

Stripping buffer

25 ml Tris-HCl, pH 6.7

↓105

20 ml 20% SDS

1.4 ml 14.3 M β-Mercaptoethanol

H2O was added to 200 ml

2.2.7 S100A14 Antibody Generation

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The anti-S100A14 polyclonal antibody was raised by immunizing two rabbits with the NH2 – CEAAKSVKLERPVRGH – COOH peptide located in the C-terminus of S100A14 protein (Eurogentec, Belgium). Animals were immunized four times at 2weeks intervals by subcutaneous injection of 50-100 μg of peptide in an emulsion with KLH. The titer of the antiserum was determined by ELISA using 100 ng of the antigen peptide. A dilution of maximally 1:1000 (antiserum SA1349) is possible for the detection of 4 μg of the antigen peptide. The antibody was affinity-purified with the antigen peptide using standard methods (Eurogentec, Belgium).

Potential cross-reactivity of the antibody with other S100 family members was examined by Western blot using recombinant S100 proteins: S100A1, S100A2, S100A3, S100A4, S100A5, S100A6, S100A8, S100A9, S100A12, S100A13, S100B, and a protein extract from HBE cells as a positive control. The recombinant proteins were kindly provided by Prof. Dr. C. Heizmann (University of Zürich, Switzerland) and Dr. C. Kerkhoff (University of Münster, Germany). No cross-reactivity with any S100 protein tested was detected confirming that the Cterminal peptide used for rabbit immunisation is indeed S100A14-specific (data not shown).

2.2.8 Immunofluorescence Analysis

For immunofluorescence analysis, cells were seeded and grown on 18 x 18 mm glass cover slips. After fixation with freshly prepared 3% paraformaldehyd in 1 x PBS for 15 min at room temperature, cells were permeabilized with 0.2% Triton X-100 for 2 min, washed 3 x 5 min with 1 x PBS and incubated for 1 hour in blocking buffer (10% FCS in 1 x PBS).

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The S100A14 affinity-purified antibody was diluted 1:2000, and anti-V5 antibody 1:200 in blocking buffer. Cover slips were incubated with the primary antibodies for 2 hours at room temperature, then washed 3 x 5 min in PBS, and incubated with the secondary antibodies for 1 hour. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (H + L) antibody at 1:200 and FITC-conjugated goat anti-rabbit antibody at 1:200 were used. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) (Sigma, Steinheim, Germany) for 5 min. Cover slips were then washed with PBS, air-dried, and mounted on glass slides with DABCO anti-fade reagent (Merck, Darmstadt, Germany). They were examined using Leica confocal laser-scanning microscope (Jena, Germany).

2.2.9 Immunohistochemistry

Immunohistochemical staining of tissue sections was performed by Ms. B. Thews, Ms. K. Witkowski, and Ms. K. Petri at the Immunohistochemistry Laboratory of the Institute of Pathology, Charité in Berlin.

Paraffin-embedded archive material was retrieved from the files of the Institute of Pathology of the Charité University Hospital. One-µm sections were cut from the blocks, mounted on superfrost slides (Menzel-Gläser, Germany) or ChemMate Capillary Cap Microscope slides (DAKO), dewaxed with xylene and gradually hydrated. Antigen retrieval was performed by pressure cooking in 0.01 M citrate buffer for 5 min. The primary antibody was incubated at room temperature for 30 min. The biotinylated secondary antibody reagent of the ChemMate Detection Kit (DAKO, Denmark), that reacts equally well with rabbit and mouse immunoglobulins, was applied as recommended by the supplier. Detection took place by indirect streptavidin-biotin method with alkaline phosphatase as the reporting enzyme according to the manufacturer’s instructions. Chromogen-Red (ChemMate Detection Kit) served as chromogen, afterwards the slides were briefly counterstained with haematoxylin and aquaeously mounted.

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Primary antibodies, corresponding secondary antibodies, and their dilutions

Primary antibody

Dilution

Secondary antibody

Anti-S100A14

1:1000

ChemMate biotinylated goat anti-rabbit

Anti-ERBB2

1:1000

ChemMate biotinylated goat anti-rabbit

Anti-Ki-67

1:1000

ChemMate biotinylated goat anti-mouse

Anti-ER

1:100

ChemMate biotinylated goat anti-mouse

Anti-PR

1:500

ChemMate biotinylated goat anti-mouse

Specificity of the S100A14 antibody was verified by incubating tissue sections with pre-immune serum, demonstrating completely negative staining.

↓109

Immunostained slides were analysed under a Zeiss Axioskop light microscope and scored after the entire slide had been evaluated. The immunohistochemical evaluation was performed by Prof. Dr. I. Petersen.

In order to analyse the expression level of S100A14, we introduced a semiquantitative scoring system consisting of four different staining patterns to distinguish between positive and negative staining. The highest positive score has been assigned to score 3, where the entire tissue shows strong positive staining. Moderately positive staining represents score 2. Score 1 was assigned to borderline positive staining of the protein and score 0 represents the complete negative staining ( Fig. 2 ).

Fig.   2 The scoring system used for immunohistochemical analysis of S100A14. Examples shown are lung tumour tissue spots stained with anti-S100A14 antibody. The four different scores used are indicated.

↓110

ERBB2 immunostaining was evaluated using the method employed by the HercepTest (DAKO) according to the degree and the proportion of plasma membrane staining. ERBB2 expression was negative for a score of 0 to 1+. A score of 0 was defined as no staining or plasma membrane staining in less than 10% of tumour cells. A score of 1+ comprised faint or partly stained plasma membrane in more than 10% of tumour tissue. Tumours with scores of 2 or greater were considered to be positive for ERBB2 overexpression. A score of 2+ was defined as weak to moderate complete plasma membrane staining in more than 10% of tumour cells. A score of 3+ was interpreted as strong, complete plasma membrane staining in more than 10% of tumour cells.

2.2.10 Tissue Microarrays (TMA) Generation

Tissue microarrays containing normal human tissues, as well as breast and lung tumours were generated by Dr. Y. Yongwei and Ms. N. Deutschmann. The normal human tissue array consisted of 29 tissue samples, the lung tumour array consisted of 150 tissue samples, and the breast tumour array was composed of 146 tissue samples. All the tissue samples were derived from randomly selected patients who underwent surgical resections performed at the Department of Surgery, University Hospital Charité between 2000 and 2002.

Suitable areas for tissue retrieval were marked on standard haematoxylin-eosin (HE) sections. Then, tissue cylinders with a diameter of 1.0 or 0.6 mm were punched out of the paraffin block and transferred into a recipient array block using a manual tissue arrayer purchased from Beecher Instruments (Woodland, USA). The tissue array was cut in 1-µm sections without any sectioning aid like adhesive tapes or additionally coated slides.

↓111

Limitations of the tumour material and slide damages were the main reasons for the missing specimens on the tissue microarray slides. Tissue spots with heterogeneous expression patterns for S100A14 were subsequently examined by whole tissue section immunohistostaining.

2.2.11 Statistical Analysis

The Fisher’s exact test for categorical variables was used to compare immunohistochemical results for S100A14 with other immunohistochemical markers and clinicopathologic characteristics. All analysis were two-tailed, and the criterion of significance was set at p < 0.05. The statistical analysis was undertaken using the SPSS statistical package (Version 11.5).

2.2.12 Bioinformatics

Blast analysis was performed to search for homologies at the nucleotide level and for pairwaise sequence alignments (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/BLAST). Recognition sites for restriction enzymes were identified with the Webcutter program at the Web address http://www.firstmarket.com/cutter/cut2.html. S100A14 promoter region was analysed by TRANSFAC 4.0 (http://www.transfac.gbf.de/TRANSFAC), using the MatInspector V2.2 program. Parameters for MatInspector were set for 1.0 core similarity (a 4-nt highly conserved sequence) and 0.85 matrix similarity, employing the vertebrates matrix group. ClustalW 1.8 program was used for multiple alignments of DNA sequences (http://www.ebi.ac.uk).

↓112

The ExPASy nucleotide translation tool (Swiss Institute of Bioinformatics, http://www.expasy.ch/tools/dna.html) was applied to translate cDNA in all six possible open reading frames. Sequence homologies at the amino acid level were determined with the EMBnet-CH Blast program and potential posttranslational modification sites with the PROSITE program (all the programs are available at the ExPASy Molecular Biology Server). Hydrophobicity analysis based on the method of Kyte and Doolittle was performed using the program ProtScale at the ExPASy Molecular Biology Server. Protein secondary structure was predicted with the PSIpred V2.0 (University College London, UK http://bioinf.cs.ucl.ac.uk/psipred).


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