Quantitative Proteome Analysis of Alveolar Type-II Cells Reveals a

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Quantitative Proteome Analysis of Alveolar Type-II Cells Reveals a Connection of Integrin Receptor Subunits Beta 2/6 and WNT Signaling Regina T. Mukhametshina,†,○ Aaron Ruhs,‡ Indrabahadur Singh,† Diya Hasan,† Adriana Contreras,† Aditi Mehta,† Vandana S. Nikam,§ Katrin Ahlbrecht,§ Gianni Carraro,⊥ Hector A. Cabrera-Fuentes,#,○ Dongsheng Jiang,▽ Robert Voswinckel,§ Werner Seeger,§ Saverio Bellusci,⊥ Karin Scharffetter-Kochanek,▽ Tatyana V. Bagaeva,○ Klaus T. Preissner,# Thomas Boettger,∥ Thomas Braun,∥ Marcus Krüger,‡ and Guillermo Barreto†,* †

LOEWE Research Group Lung Cancer Epigenetic, ‡Division of Biomolecular Mass Spectrometry, §Department of Lung Development and Remodeling, and ∥Department of Cardiac Development and Remodeling, Max-Planck-Institute for Heart and Lung Research, member of the Universities of Giessen and Marburg Lung Center (UGMLC) and German Center of Lung Research (DZL), Parkstraße 1, 61231 Bad Nauheim, Germany ⊥ Chair for Lung Matrix Remodeling, Excellence Cluster Cardio Pulmonary System and #Biochemistry Institute, Medical School, Justus-Liebig-University, Friedrichstrasse 24, 35932 Giessen, Germany ▽ Department of Dermatology and Allergic Diseases, Ulm University, Albert-Einstein-Allee 23, 89081 Ulm, Germany ○ Kazan (Volga Region) Federal University, 18 Kremlyovskaya St., Kazan 420008, Russian Federation S Supporting Information *

ABSTRACT: Alveolar type-II cells (ATII cells) are lung progenitor cells responsible for regeneration of alveolar epithelium during homeostatic turnover and in response to injury. Characterization of ATII cells will have a profound impact on our understanding and treatment of lung disease. The identification of novel ATII cell-surface proteins can be used for sorting and enrichment of these cells for further characterization. Here we combined a high-resolution mass spectrometry-based membrane proteomic approach using lungs of the SILAC mice with an Affymetrix microarraybased transcriptome analysis of ATII cells. We identified 16 proteins that are enriched in the membrane fraction of ATII cells and whose genes are highly expressed in these cells. Interestingly, we confirmed our data for two of these genes, integrin beta 2 and 6 (Itgb2 and Itgb6), by qRT-PCR expression analysis and Western blot analysis of protein extracts. Moreover, flow cytometry and immunohistochemistry in adult lung revealed that ITGB2 and ITGB6 are present in subpopulations of surfactant-associated-protein-C-positive cells, suggesting the existence of different types of ATII cells. Furthermore, analysis of the Itgb2−/− mice showed that Itgb2 is required for proper WNT signaling regulation in the lung. KEYWORDS: ATII cells, integrin, WNT signaling, SILAC mice, lung

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INTRODUCTION

alveoli. ATII cells are responsible for regeneration of alveolar epithelium during homeostatic turnover and in response to injury.4−7 Characterization of the regulatory mechanisms controlling the proper balance between expansion and differentiation of ATII cells will have a profound impact on our understanding of lung regeneration and treatment of lung disease. However, detailed characterization of ATII cells has been challenging because the most specific known cellular

The lung is a complex organ consisting of different epithelial and mesenchymal cell lineages organized in a proximal-distal manner, with several specialized cell types that form the functional gas exchange interface required for postnatal respiration (Figure 1).1,2 The lung shows slow homeostatic turnover but rapid repair after injury, and tissue-resident lungendogenous progenitor cell niches located in specific regions along the proximal−distal axis of the airways are thought to be responsible for both processes.3 ATII cells represent one of these regional progenitor cell populations and are located in the © 2013 American Chemical Society

Received: June 17, 2013 Published: October 31, 2013 5598

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extracellular region, a single-pass transmembrane domain, and a short cytoplasmic tail.11 Integrins mediate cell−cell and cell− ECM (extracellular matrix) interactions and transmit signals across the plasma membrane in both directions between their extracellular ligand binding adhesion sites and their cytoplasmic domain,12,13 thereby linking the cytoskeleton to several signal transduction pathways.14−18 Further analysis of the membrane fraction based on Gene Ontology terms showed an enrichment of proteins that are involved in WNT signaling. Characterization of the lung of Itgb2−/− mice19 revealed that Itgb2 seems to be required for a negative regulation of WNT signaling. Our work thereby provides the basis for further studies that may decipher the regeneration potential of ATII cells in health and disease.

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EXPERIMENTAL SECTION

Cell Culture

Primary alveolar type II cells (ATII cells) were isolated from C57BL/6 mice, as previously described, 20 with minor modifications. Crude cells suspensions from the lungs were prepared by intratracheal instillation of agarose containing dispase (BD Heidelberg, cat. no. 354235), followed by mechanical disaggregation of the lungs. Crude cell suspensions were purified by negative selection using a system consisting of biotinylated antibodies (Biotin antimouse CD16/CD32, cat. no. 553143; Biotin antimouse CD45 (30-F11), cat. no. 553078, both from BD Biosciences), streptavidin-coated magnetic beads (Promega, cat. no. Z5481), and a magnetic separator stand (Promega, cat. no. Z5410). Purified ATII cells were seeded on fibronectin-coated cell-culture dishes and cultured up to 3 days in D-MEM/F-12 (1:1) (Life Technologies, cat. no. 31330038) supplemented with 10% FCS and 1% penicillin/streptomycin (Pen/Strep, Gibco, 15070) in an atmosphere of 5% CO2 at 37 °C. We obtained culturable ATII cells from C57BL/6 mice at a purity of 86.3% ± 5.2 (mean ± SD; n = 3) and total yields of 4.7 ± 0.5 × 106 cells per mouse (Suppl. Figure 1 in the Supporting Information). Mouse epithelial lung cells (MLE-12, ATCC CRL-2110), mouse normal lung cells (MLg, ATCC CCL-206), and mouse fibroblast (NIH/3T3, ATCC CRL-1658) were obtained from the American Type Culture Collection. Mouse fetal lung mesenchyme cells (MFLM-4) were obtained from Seven Hills Bioreagents. All cell lines were cultured following the supplier instructions. MLE-12 cells were transiently transfected with Itgb2-YFP expression plasmid (Addgene, cat. no. 8638) using Lipofectamine 2000 transfection reagent (Invitrogen) at a ratio of 1:2 of DNA/lipofectamine according to the manufacturer instructions. Cells were harvested 48 h after transfection for further analysis. Where indicated, MLE-12 cells were treated with lithium chloride (LiCl, 20 mM for 8 h) to activate the WNT signaling pathway.

Figure 1. Schematic representation of the lung structure. The lung consists of different structural regions organized along a proximaldistal axis. Each of these regions is characterized by specialized cell types of epithelial or mesenchymal origin (listed in the square). Different tissue-resident lung-endogenous progenitor cells (underlined in the list) are located in specific regions along the proximal−distal axis of the airways. They are responsible for homeostatic turnover and repair after injury. Alveolar type II (ATII) cells represent one of these regional progenitor cell populations and are located in the alveoli. Sm Mus, smooth muscle cells; Clarav, variant Clara cells; PNEC, pulmonary neuroendocrine cells; BASC, bronchio-alveolar stem cells; AT I, alveolar type-I cells.

marker for these cells (surfactant-associated protein C, SFTPC, or SP-C) is a secreted molecule, making the enrichment of a homogeneous population of these cells difficult to attain. The aim of our study was to find new cell-surface proteins that are specific for ATII cells and can be used for sorting and enrichment of ATII cells for further characterization. Metabolic labeling of living organisms with stable isotopes has become a powerful tool for global protein quantification. The SILAC (stable isotope labeling with amino acids in cell culture) approach is based on the incorporation of nonradioactive-labeled isotopic forms of amino acids into cellular proteins.8 The effective SILAC labeling of immortalized cells and single-cell organisms (e.g., yeast and bacteria) was recently extended to more complex organisms, including worms, flies, and even rodents.9 The administration of a 13C6-lysinecontaining (Lys6-heavy) diet for one mouse generation leads to a complete exchange of the natural isotope 12C6-lysine (Lys0-light). Here we used the lung of the fully labeled SILAC mice as a heavy “spike-in” standard into nonlabeled samples of murine ATII or MLE-12 cells (mouse lung epithelial cell line) in combination with high-performance mass spectrometry to analyze fractions of membrane proteins. A comparison of the results obtained by the proteomic approach with an Affymetrix microarray-based expression analysis of ATII cells led us to the identification of 16 membrane proteins that are highly expressed and present in ATII cells. We focused our attention on two of these proteins, integrin receptor subunits beta 2 and 6 (ITGB2 and ITGB6), and confirmed that they are indeed present in ATII cells. Integrins are heterodimeric cell adhesion molecules that are formed by specific noncovalent association of an alpha and a beta subunit.10 In general, each integrin subunit has a large

Animal Experiments

C57BL/6 mice (stock no. 002644, Jackson Laboratories)21 were obtained from Charles River Laboratories at 5 to 6 week of age. Integrin-β2-deficient mice (Itgb2−/−, B6.129S7Itgb2tm2Bay/J, stock no. 003329,19) were obtained from the animal care facility of the University of Ulm. Animals were housed and bred pathogen-free under controlled temperature and lighting (12/12 h light/dark cycle) and fed with commercial animal feed and water ad libitum. All experiments were performed with 6−8 week old mice in compliance with the German Law for Welfare of Laboratory Animals(§115599

dx.doi.org/10.1021/pr400573k | J. Proteome Res. 2013, 12, 5598−5608

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For data analysis, we used the MaxQuant software tool,25 version 1.0.14.10. The measured raw data were processed and quantified as described.26 For Gene Ontology functional analysis of the data, the GORILLA online tool was used in target and background mode for ATII-enriched proteins (Eden, BMC Bioinformatics, 2009).

Genehmigung; IVMr46-53r30.03.MPP04.12.02; IVMr4653r30.03.MPP06.12.01) and according to the institutional guidelines that comply with national and international regulations. The lungs of wild type and Itgb2−/− mice were harvested and used for RNA isolation, protein isolation, or immunohistochemistry. Metabolic labeling of living C57BL/6 mice was achieved by a diet containing a non-radioactive-labeled isotopic form of the amino acid lysine 13C6-lysine (Lys6 - heavy). The administration of a heavy lysine-containing diet for one mouse generation leads to a complete exchange of the natural isotope 12 C6-lysine (Lys0 -light) in the cellular proteins. The fully labeled SILAC mice were used as a heavy “spike-in” standard into nonlabeled ATII- or MLE-12 cells samples during global proteomic screening with high-performance mass spectrometers.

Affymetrix Microarray Transcriptome Analysis

Total RNA was extracted from MLE12 cells using the guanidinium isothiocynate method (TRIzol reagent; Invitrogen, Carlsbad, CA). RNA quality was assessed by using the Agilent Model 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). For mRNA expression analysis, the Affymetrix GeneChip Mouse Gene 1.0 ST Array was used with the respective once-cycle target-labeling protocol according to the manufacturer’s instructions. Data were analyzed by the RMA algorithm using the Affymetrix Expression Console. An unpaired t test was performed with log2-transformed data to identify significantly differentially expressed transcripts. Fold change was calculated using DNAStar ArrayStar 3.0 software. The ATII cell transcriptome analysis was previously described,27 and the database is available in the Gene Expression Omnibus (GEO) under the accession number GSE6846.

Membrane Protein Isolation

ATII cells or MLE-12 cells were mixed with lung tissue from SILAC mice at a ratio of 1:1 (18 mg/18 mg; wet weight/wet weight). The membrane proteins of these mixtures were isolated as previously described.22,23 In brief, the mixtures were homogenized in 1 mL of 2 M NaCl, 10 mM HEPES/NaOH, pH 7.4, and 1 mM EDTA. All steps were performed in the presence of protease inhibitor cocktail “Complete” (Roche) at 4 °C. The crude homogenates were centrifuged in a benchtop centrifuge at 1600g for 20 min. The pellets were rehomogenized in 1 mL of 0.1 M Na2CO3, 1 mM EDTA, pH 11.3 to remove nonmembrane-soluble proteins. After a second centrifugation step, the pellets were extracted with 5 M urea, 100 mM NaCl, 10 mM HEPES, pH 7.4, and 1 mM EDTA to further remove nonintegral membrane proteins that were previously unaffected by the carbonate treatment. After two washing steps with 0.1 M Tris/HCl, pH 7.6, the pellets containing the membrane protein fractions were solubilized in 0.1 M TRIS/HCl pH 7.6 containing 2% SDS and 50 mM DTT at 90 °C for 1 min.

Semiquantitative and Quantitative RT-PCR

Total RNA was isolated with RNeasy plus mini kit (Qiagen). cDNA was synthesized from total RNA using the high-capacity cDNA reverse transcription kit (Applied Biosystem) according to manufacturer’s instructions. The PCR results were normalized with respect to the housekeeping gene Gapdh. Quantitative real-time PCR reactions were performed using SYBR green on the step one plus real-time PCR system (Applied Biosystem). Western Blot

Protein concentrations were determined using BCA kit (Sigma). Western blot was performed using standard methods.28 Immunodetection of blotted proteins was performed using ITGB2-, ITGB6-, CD14-, CD45-, AXIN2-, BMP4-, MYCN-, ABC- (all from Millipore), and LMNB1(Santa Cruz) specific primary antibodies, the corresponding HRP-conjugated secondary antibodies, an enhanced chemiluminescent substrate (SuperSignal West Femto, Thermo Scientific), and a luminescent image analyzer (Las 4000, Fujifilm).

Mass Spectrometry: Sample Preparation, Methods, and Data Analysis

Solubilized membrane protein fractions were prepared for proteome analysis by FASP (filter-aided sample preparation) as previously described.24 Reverse-phase nano-LC−MS/MS was performed by using an Agilent 1200 nanoflow LC system (Agilent Technologies, Santa Clara, CA) using a cooled thermostatted 96-well autosampler. The LC system was coupled to an LTQ-Orbitrap Velos instrument (Thermo Fisher Scientific) equipped with a nanoelectro-spray source (Proxeon, Denmark). Chromatographic separation of peptides was performed in a 10 cm long and 75 μm C18 capillary needle. The column was custommade with a methanol slurry of reverse-phase ReproSil-Pur C18-AQ 3 μm resin (Dr. Maisch). The peptide mixtures were loaded onto the column with a flow rate of0.5 μL/min and then eluted with a linear gradient at a flow rate 0.25 μL/min. The mass spectrometer was operated in the data-dependent mode to automatically measure MS and MS/MS spectra. LTQ-FT full scan MS spectra (from m/z 350 to 1750) were acquired with a resolution of r = 60,000 at m/z = 400. The five most intense ions were sequentially isolated and fragmented in the linear ion trap by using collision-induced dissociation with collision energy of 35%. Further mass spectrometric parameters: spray voltage of 2.4 kV, no sheath gas flow, dynamic exclusion was set to 120 s, and capillary temperature was 200 °C.

Flow Cytometry Analysis of a Single-Cell Suspension of the Lungs

Lung single-cell suspensions were generated and analyzed by flow cytometry, as previously described,29 with minor modifications. Primary antibodies used were Pro-SFTPC (Millipore), ITGB2-CD18/APC (BioLegend, 0.5 mg/mL), and ITGB6/FITC (R&D). Secondary antibodies used were Alexa 488 (BIOTIUM) and Alexa 633 (BIOTIUM). After immunostaining, single-cell suspensions were analyzed using the BD LSRII flow cytometer. Data were analyzed with the BD FACS DIVA Software Version 6.1.3. Immunohistochemistry

For cryosections, mouse lungs were harvested and embedded in tissue-freezing medium (Polyfreeze, Polysciences). Sections of 10 μm were prepared on a cryostat (Leica Germany) and postfixed in 4% PFA for 20 min. Antibody staining was performed following standard procedures. The sections were 5600

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Figure 2. (A) Schematic representation of experimental procedure. Spike-in -based relative quantification of ATII versus MLE-12 cells using 13C6lysine labeled lung (Lys-6-labeled, heavy labeled lung) as standard. ATCC, American Type Culture Collection. (B) Quality analysis of membrane protein isolation. Distribution of Gene Ontology cellular component (GOCC) terms-based analysis of identified proteins after mass spectrometric measurement. (C) Calculation of direct abundance ratio between MLE-12 and ATII cells (MLE-12/ATII). (D) (Top) Histogram of spike-in SILAC-ratios (log2) between heavy labeled lung and ATII (left) or MLE-12 (right) cells. (Bottom) Histogram of direct ratio between MLE-12 versus ATII cells (MLE-12/ATII, lo 2, left) and the direct ratio plotted against intensity (log10, right).

Statistical Analysis

examined with a Zeiss confocal microscope (Zeiss, Germany). Antibodies used were specific against Pro-SFTPC (Millipore), ITGB2/CD18 (R&D system), and ITGB6 (R&D system). Secondary antibodies used were Alexa 488 and Alexa 594 (Invitrogen). DRAQ5 (eBioscience) was used as nuclear dye. For paraffin-embedded mouse lung tissue, lungs were postfixed overnight in 1% PFA at 4 °C, dehydrated over a graded series of alcohol, and paraffin-embedded. Sections of 4 μm were prepared on a microtome (Leica Germany). Antigen retrieval was performed by cooking using a rice-cooker for 20 min in citrate buffer containing 10 mM sodium citrate, 0.05% Tween 20, pH 6.0. Antibody staining was performed following standard procedures. Primary antibody used was specific against activated β-CATENIN (Millipore). The sections were examined with a Zeiss confocal microscope (Zeiss Germany).

Statistical analyses were performed using Excel Solver. All data are represented as mean ± standard error (mean ± s.e.m). One-way analyses of variance (ANOVA) were used to determine the levels of difference between the groups and P values for significance. P values after one-way ANOVA:* p ≤ 0.05; ** p < 0.01, and *** p < 0.001

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RESULTS

Mass Spectrometry Analysis of Membrane Proteins of ATII and MLE-12 Cells

Figure 2A shows a schematic representation of the experimental workflow. Primary ATII cells from adult mouse lung were isolated and cultured as previously described.20 Nonlabeled ATII cells or MLE-12 cells were mixed with lung tissue from 5601

dx.doi.org/10.1021/pr400573k | J. Proteome Res. 2013, 12, 5598−5608

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Figure 3. (A) Table of selected proteins enriched in ATII cells (first column). Direct abundance ratios between lung tissue versus ATII cells (fourth column), lung tissue versus MLE-12 cells (seventh column), and MLE-12 versus ATII cells (eighth column) are represented. Values taken for calculation were normalized to heavy lung tissue. The numbers of peptides and unique peptides used for the calculations are presented in the other columns. Only high-confidence peptides were considered for the calculation. H, heavy; L, light; no., number; n.d., not determined. (B) MS spectra of ITGB2 specific SILAC-pairs derived from ATII or MLE-12 cells mixed with labeled heavy lung. m/z, mass by charge ratio.

reticulum, and golgi apparatus, a cellular organelle with a high content of endomembrane that is particularly important in the processing of membrane proteins and proteins for secretion. Interestingly, over 90 proteins with the GOCC term plasma membrane also match the terms signaling and receptor. These results support the high efficiency of our membrane protein fractionation approach. After processing the mass spectrometric raw data, we calculated the direct Ratio MLE-12/ATII by the ratio-by-ratio method (Figure 2C). Dividing the heavy lung/ATII cells ratio (ratio 1) by the heavy lung/MLE-12 cells ratio (ratio 2) will cancel out the heavy lung and results in the direct ratio MLE12/ATII. For 1387 proteins we observed enriched levels in ATII cells, whereas 618 proteins showed increased abundance in MLE-12 cells. The distribution of SILAC ratio between the SILAC lung tissue and the nonlabeled cell lines is shown in

SILAC-mice at a ratio of 1:1 (wet weight/wet weight). By measuring the Lys-6-labeled lung tissue alone, we observed an average incorporation rate of ∼97% (Suppl. Figure 2 in the Supporting Information). Membrane proteins of these mixtures were isolated,22,23 and after adequate sample preparation,24 they were analyzed by high-resolution mass spectrometry-based proteomic approach. We identified more than 6000 proteins in lung tissue, ATII, and MLE-12 cells. To check the enrichment of membrane proteins, we performed a Gene Ontology cellular component (GOCC) terms-based analysis of the data set using the GORILLA online-tool (Eden, BMC Bioinformatics, 2009) and found the GOCC terms membrane and plasma membrane to be highly enriched (Figure 2B). This GOCC terms analysis revealed that >90% of the proteins identified after mass spectrometric analysis are potentially localized to membranes, including plasma membrane (∼4000 proteins), endoplasmic 5602

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Figure 4. Identification of potential ATII cell-specific membrane proteins. (A) Scatter plot between membrane protein abundance ratio (MLE-12/ ATII) and gene expression ratio (MLE-12/ATII). Proteins enriched in the membrane of ATII cells are indicated in the marked section and listed in the table (B). Prot Abud, protein abundance; Gene Exp, gene expression.

cells. Conversely, we identified 124 candidates with increased protein levels and no change on mRNA level between ATII and MLE-12 cells.

Figure 2D. For 1388 candidates, we identified only signals from the labeled tissue, and conversely 118 candidates were only observed in MLE-12/ATII cells. We selected 28 proteins from the membrane proteomic data set and presented them in the Table of Figure 3A with spike-in ratios, direct MLE-12/ATII ratios and peptide counts. From this group of proteins, 26 proteins are enriched in ATII cells when compared with complete lung (second column; Ratio H/L Lung versus ATII < 1). Moreover, 23 proteins were specifically enriched in ATII cells when compared with MLE-12 cells (first column; Fold change ≤ 0.07). The ATII cellular marker SFTPC was slightly elevated in ATII cells when compared to MLE-12 cells, whereas two of the selected proteins (EPHA2 and CD9) showed similar levels in both cell types analyzed. The spectra of two ITGB2 specific peptides (Figure 3B) demonstrate that ITGB2 is highly enriched in ATII cells when compared with the whole lung spike-in (left panel) and almost nonexistent in MLE-12 cells (right panel).

Integrin Receptor Subunits Beta 2 and 6 Are Membrane Proteins of a Subpopulation of Alveolar Type-II Cells

Next, we focused our attention on ITGB2 and ITGB6 for further analysis to confirm our results from the membrane proteome and transcriptome analyses. The expression of Itgb2 and Itgb6 was determined in MLg (mouse normal lung cells), MFLM-4 (mouse fetal lung mesenchyme cells), NIH/3T3 (mouse fibroblasts), MLE-12, and ATII cells as well as in adult mouse lung by quantitative PCR after reverse transcription (qRT-PCR) (Figure 5A). Itgb2 and Itgb6 are highly expressed only in ATII cells when compared to the other cell lines tested. In contrast, Cox2 expression, another gene that was identified in the membrane proteome approach, was detected not only in ATII but also in MLg and MFLM-4 cells. Sf tpc was expressed in ATII and MLE-12 cells, as expected. Consistently, Western blot analysis of protein extracts (Figure 5B) showed that the level of ITGB2 and ITGB6 was high in spleen, lung, and ATII cells but not in any of the other cell lines tested. Our results support cellular specificity for the expression of Itgb2 and Itgb6. Because ITGB2 (CD18) is known to be expressed together with various integrin alpha subunits in blood cells,30,31 we considered the possibility that our data may be the result of putative contamination of the isolated ATII cells with remaining blood cells. Therefore, we tested the presence of two typical blood cell membrane proteins, CD14 and CD45,32−34 in our protein extracts. As expected, CD14 and CD45 are present in the spleen protein extract but absent in any of the other protein extracts analyzed, ruling out the possibility that our results are due to a contamination of the isolated ATII cells with remaining blood cells

Identification of Potential ATII Cell-Specific Membrane Proteins

The transcriptomes of ATII and MLE-12 cells were determined by microarray-based expression analysis (Affymetrix). The transcriptome as well as the membrane proteome databases of both cell types were cross-analyzed (Figure 4A) by calculating the ratios of expression of the genes in MLE-12 versus ATII cells and comparing them with the respective protein ratios. This integrative approach led to the identification of 16 genes that were highly expressed in ATII cells and whose gene products were enriched in the membrane of ATII cells (Figure 4B). Thus, the identified genes are potential ATII cell-surface markers. Interestingly, we also detected 226 candidates that were enriched only on the mRNA level more than two-fold and showed a similar protein distribution (