2 Materials and Methods

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.

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 I. Petersen on tumorigenicity of various human cultured lung cancer cell lines in nude mice (unpublished results).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.

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

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).

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

1.80 g KH₂PO₄

0.45 g Na-citrate*2H₂O

0.09 g MgSO₄*7H₂O

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.

1 x LB broth

1% Bacto tryptone

0.5 % Bacto yeast extract

1% NaCl

1 x LB agar

LB broth with 1.5% Bacto agar

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).

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.

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:

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

2 g/l KCl

2 g/l KH₂PO₄

21.6 g/l Na₂HPO₄*7 H₂O

pH was adjusted to 7.4

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.

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”.

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.

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

140 mM NaCl

5 mM KCl

1 mM MgCl₂

10 mM Glucose

10 mM HEPES

1 mM CaCl₂

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.

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.

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.

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.

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

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.

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.

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.

Amplification of DNA was performed according to the following protocol:

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:

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.

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.

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.

Gel components were pre-mixed the following way:

30 ml Sequagel XR (Biozym)

7.5 ml Sequagel-buffer (Biozym)

150 µl 10% Ammonium persulfate (APS)

15 µl TEMED

and polymerised for 1 hour.

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

4 µl plasmid DNA (100-300 ng/µl)

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

to a total reaction volume of 12 µl.

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:

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.

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.

10 x TBE

108 g Tris-base

55 g Boric acid

40 ml 0.5 M EDTA, pH 8.0

adjusted with H₂O to 1 l

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.

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.

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.

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

0.3 M Natrium citrate

20 x SSPE

3.6 M NaCl

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.

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).

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).

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.

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.

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.

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.

DEPC-treated H ₂ O

1 l H₂O

1 ml Diethylpyrocarbonate (DEPC)

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

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:

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

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.

10 x MOPS

0.2 M MOPS

50 mM Sodium acetate

10 mM EDTA, pH 8.0

pH was adjusted to 7.0 with NaOH

10 x Gel-loading buffer

500 μl Formamide

2 μl EDTA, pH 8.0

5 μl Ethidium bromide

0.1% w/v Bromophenol blue

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.

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.

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.

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.

Denaturing hypotonic lysis buffer

1% SDS

10 mM Tris-HCl, pH 7.5

2 mM EDTA, pH 8.0

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.

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.

Hypotonic lysis buffer

10 mM Tris-HCl, pH 8.0

0.1 mM DTT

1 mM PMSF

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

2 x SDS sample buffer

120 mM Tris-HCl, pH 6.8

200 mM DTT

4% w/v SDS

10% w/v Glycerol

0.002% w/v Bromophenol blue

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

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.

Destaining solution

90 ml Methanol

2 ml Acetic acid

8 ml H₂O

Elution solution

50 ml Ethanol

10 μl 0.5 M EDTA, pH 8.0

0.5 ml 5 M NaOH

49.5 ml H₂O

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

3.35 ml H₂O

2.5 ml 1.5 M Tris-HCl, pH 8.8

4.0 ml 30% Acrylamide/Bisacrylamide

100 μl 10% SDS

50 μl APS

10 μl TEMED

Stacking gel: 5 ml of 4% gel

3.05 ml H₂O

1.25 ml 0.5 M Tris-HCl, pH 6.8

0.66 ml 30% Acrylamide/Bisacrylamide

50 μl 10% SDS

50 μl APS

10 μl TEMED

5 x running buffer

15.1 g/l Tris-base

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.

2 x Tricine sample buffer

2.0 ml 0.5 M Tris-HCl, pH 6.8

2.4 ml Glycerol

4.2 ml 20% SDS

0.31 g DTT

0.02% w/v Coomassie blue

adjusted with H₂O to 10 ml

Separating gel: 10 ml of a 15% gel

1.66 ml H₂O

3.32 ml 3 M Tris-HCl, pH 8.45

1.06 ml Glycerol

3.75 ml 40% Acrylamide/Bisacrylamide

150 μl 20% SDS

50 μl APS

10 μl TEMED

Stacking gel: 5 ml of 4% gel

2.71 ml H₂O

1.66 ml 3 M Tris-HCl, pH 8.45

0.5 ml 40% Acrylamide/Bisacrylamide

75 μl 20% SDS

50 μl APS

10 μl TEMED

Cathode electrophoresis buffer

12.11 g/l Tris-base

17.92 g/l Tricine

1 g/l SDS

stored at 4°C

Anode electrophoresis buffer

24.22 g Tris-base

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

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.

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

Transfer buffer

5.81 g Tris-base

2.93 g Glycine

1.875 ml 20% SDS

200 ml Methanol

H₂O was added to 1 l

TBST

10 ml 1 M Tris-base, pH 8.0

30 ml 5 M NaCl

500 μl Tween-20

H₂O was added to 1 l

Coomassie blue staining solution

50% v/v Methanol

0.05% v/v Coomassie blue R-250

10% v/v Acetic acid

40% H₂O

Coomassie blue destaining solution

5% v/v Methanol

7% v/v Acetic acid

88% H₂O

Blocking buffer

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

Stripping buffer

25 ml Tris-HCl, pH 6.7

20 ml 20% SDS

1.4 ml 14.3 M β-Mercaptoethanol

H₂O was added to 200 ml

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).

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).

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).

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.

Primary antibodies, corresponding secondary antibodies, and their dilutions

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

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.

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.

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.

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.

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).

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).

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).