In this study, sections from tissue microarrays (TMA) have been used. TMAs from archived tissue specimens were constructed and used upon approval by the Ethics Committee of Basel (Ethikkommission beider Basel, EKBB, www.ekbb.ch
) on November 3rd
, 2003. According to the approval, a written informed consent by the patients was not needed due to the retrospective nature of the study.
Tissue Microarrays (TMA)
TMAs were constructed as described before 
. The use of HNSCC TMA with 365, the multi-tumor TMA with 3417 and the normal TMA with 608 patient samples have been previously described 
Fluorescence In-situ Hybridization (FISH)
For Ano1 analysis, a digoxigenated FISH probe from two BAC clones (RP11-203N8 and RP11-109F24, imaGenes GmbH, Berlin, Germany) were created as described previously 
. For CCND1 analysis, a Spectrum Orange-labeled CCND1
probe was used (Vysis, Downers Grove, IL). CEP11 centromeric probes were used as reference (Vysis). Indirect labeling of the digoxigenated Ano1
FISH probe was carried out according to the ‘Fluorescent Antibody Enhancer Set for DIG Detection’ (Roche Applied Science, Rotkreuz, Switzerland). FISH signals were scored with a Zeiss fluorescence microscope and evaluated independently by CR, SS, and FR. Ano1
were defined as amplified when signals were at least twice as common as centromere 11.
For IHC, a monoclonal antibody (SP31, Cell Marque, CA) was used according to the recommended protocol and was established for the diagnostic use in GISTs. The IHC was scored using a composite scoring system: Briefly, scores were calculated by multiplying the intensity (integer between 0 and 3) with the percentage of tumor cells having this intensity. For the statistical analysis, samples with a score >0 were classified as IHC positive.
Reverse Transcriptase PCR
Total RNA was isolated from BHY, CAL-33 and CAL-27 cells using RNeasy Mini-Kit (Qiagen; Hilden, Germany). 2 µg of total RNA was reverse-transcribed in 50 µl for 1 h at 40°C using random primer and RT. 30 cycles of RT-PCR was performed using standard procedures (GoTaq DNA Polymerase, Promega), 1 µl RT and primers for anoctamis (0.5 µM, see Table S2
). For semiquantitative comparison, ß-actin was co-amplified (primer 0.05 µM). Products were analyzed on ethidium bromide-stained 2% agarose gels.
Cell Culture, siRNA and Transfection
BHY, CAL-33 and CAL-27 cell lines were grown in Opti-MEM (Gibco) supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS, Gibco) at 5% CO2 and 37°C. Lipofectamine™2000 Transfection Reagent (Invitrogen, Karlsruhe, Germany) was used for transfection of hAno1 siRNA (Stealth siRNA, Invitrogen, 5′-GGUUCCCAGCCUACCUCACUAACUU-3′, 5′-AAGUUAGUGAGGUAGGCUGGGAACC-3′) or Negative Control High GC (Invitrogen) according to the manufacture’s guidelines. Experiments were performed 48 h-72 h after transfection.
Cells were grown on glass cover slips and mounted on a perfused bath on the stage of an inverted microscope (IM35, Zeiss) and kept at 37°C. The bath was perfused continuously with Ringer solution (mM: NaCl 145, KH2
1.6, D-glucose 6, MgCl2
1, Ca-gluconate 1.3, pH 7.4) at about 5 ml/min. Patch-clamp experiments were performed in the fast whole-cell configuration as described earlier 
Cells were lysed with an appropriate buffer (150 mM NaCl, 50 mM Tris–HCl, 1 mM EDTA, 1% NP-40, protease inhibitor, 100 mM DTT; pH 7.4) and DNA was sheared by sonication. Samples were quantified using a Bio-rad protein assay (Biorad) and the same amount of protein (50 µg) was separated using PAGE (7,5%). Protein were transferred to PVDF membranes (Millipore), and probed overnight at 4°C with a rabbit monoclonal anti-ANO1 antibody (Novus Biologicals, Littleton, CO, USA). Blots were visualized using a secondary HRP-conjugated anti-rabbit antibody (Acris, Herford, Germany) and Super Signal® West Pico Chemiluminescent Substrate (Pierce, Rockford, IL, USA).
Wound Healing Assay
BHY cells were plated (5×105 cells) in µ-dishes (ibidi, Martinsried, Germany) and transfected with siRNA when 70% confluent. Confluent monolayers (48 h after transfection) were then transferred to a pre-warmed Dulbecco’s Modified Eagle Medium (DMEM, Gibco) containing D-Glucose (1000 mg/l), Sodium Pyruvate, HEPES (25 mM), and aphidicolin (5 µM), and wounded with a disposable 200 µl tip. Pictures were taken every 2 min for 24 h on an inverted microscope (Observer Z1; Carl Zeiss, Inc.) operated with the ZEN 2008 software, with a polychromatic illumination system VisiChrome (Visitron) at 37°C. Image processing was done using Adobe Photoshop CS4.
Impedance based xCELLigence Migration/proliferation Assay
The xCELLigence invasion assay (Roche, Germany) is based on changes in electrical impedance at the interphase between cell and electrode as migrating cells move through a barrier 
. These changes were directly correlated with the migrative capacity of BHY and CAL-33 cells. The technique provides an advantage over existing methods such as boyden chamber and matrigel assays, and standard proliferation assays, respectively, since the data is obtained continuously in real-time, when compared to end-point analysis in other methods. For migration assays cells were seeded at a density of 10.000 cells/well on CIM-plates-16 migration plates. For proliferation assays cells were seeded at a density of 5.000 cells/well on E16 plates.
Measurement of Intracellular Ca2+ and Cell Volume
BHY and CAL-33 cells were seeded on glass cover slips and loaded with 2 µM Fura-2/AM and 0.02% Pluronic F-127 (Invitrogen, Darmstadt, Germany) in ringer solution (mmol/l: NaCl 145; KH2PO4 0,4; K2HPO4 1,6; Glucose 5; MgCl2 1; Ca2+-Gluconat 1,3) for 1 h at room temperature. Fluorescence was detected in cells perfused with Ringer’s solution (8 ml/min) at 37°C using an inverted microscope (Axiovert S100, Zeiss, Germany) and a high speed polychromator system (VisiChrome, Puchheim, Germany). Fura-2 was excited at 340/380 nm, and emission was recorded between 470 and 550 nm using a CoolSnap camera (CoolSnap HQ, Visitron). For volume measurements cells were loaded with 1 µM calcein-AM (Invitrogen, Darmstadt, Germany) and 0.01% Pluronic F-127 (Invitrogen, Darmstadt, Germany) in ringer solution for 1 h at room temperature. Calcein was excited at 490 nm, and the emission was recorded between 520 and 550 nm. Cell swelling was induced by removing of 30 mM NaCl from ringer solution. The control isotonic solution was prepared by adding 60 mM mannitol. Data were analyzed using the software package Meta-Fluor (Universal imaging, USA) and Origin (OriginLab Corporation, USA).
Materials and Statistical Analysis
All compounds used were of highest available grade of purity and were from SIGMA. Student’s t-test (for paired or unpaired samples as appropriate), Fisher’s exact tests and ANOVA were used for statistical analysis. P<0.05 was accepted as significant. Survival curves were evaluated by the Kaplan-Meier method and log-rank test.