Supporting InformationShaykhiev et al. 10.1073/pnas.1303058110SI MethodsAirway Epithelium and Basal Cells. Samples of large airway epi-thelium (LAE) from healthy nonsmokers (n = 21) and healthysmokers (n = 31) were collected by bronchoscopic brushing ofthe third- to fourth-order bronchi as described previously (1).Subjects were recruited under a protocol approved by the WeillCornell Medical College Institutional Review Board, with writteninformed consent obtained from each volunteer before enroll-ment in the study. Cells were processed as described previously(1). Airway basal cells (BCs) were cultured from the LAE ofhealthy nonsmokers and healthy smokers as described previously(2). At day 7 or 8 of culture, when the cells were 70% confluent,the cells were removed from the plates with trypsin-EDTA. TheBC phenotype of the isolated cells was confirmed by positivestaining for BC marker keratin (KRT) 5 (>98% positive cells)and negative staining for secretory cell marker mucin (MUC)5AC, ciliated cell marker β-tubulin IV monoclonal antibody,neuroendocrine cell marker chromogranin A, and mesenchymalcell marker N-cadherin (2).

Air–Liquid Interface Model of Airway Epithelial Differentiation. Thecapacity of obtained BCs to generate differentiated progenies wasassessed by culturing on the air–liquid interface (ALI) model ofairway epithelial differentiation as described previously (2). Inbrief, after BCs reached 70–80% confluence, cells were trypsi-nized and seeded at a density of 6.0 × 105 cells/cm2 onto 0.4-μm-pore Costar Transwell inserts (Corning) precoated with type IVcollagen (Sigma-Aldrich). As soon as the cells reached conflu-ence, the apical surfaces of the cells were exposed to air (ALIday 0). The ALI medium was added from the basolateral sideevery other day up to ALI day 28, when BCs typically generatedifferentiated airway epithelium (2).

Cigarette Smoke Exposure of the Airway Epithelium In Vitro. Tomimic in vivo cigarette smoking, airway epithelium differentiatedafter 28 d of culture in ALI was exposed to cigarette smoke extract(CSE), prepared as described previously (1), from the apicalsurface by adding 1% or 2% CSE, concentrations considerednontoxic to differentiated human airway epithelial cells (1).Control cells were treated with culture medium only. The apicalmedium containing CSE remained in the upper chamber untilthe next day, when the resistance was measured and the mediumwas replaced with a fresh CSE-containing medium. The stim-ulations with CSE were carried out for 24 h every other day overa 2-wk period.After the completion of CSE exposure, supernatants were

collected from the apical and basolateral ALI compartments forELISA, and cell RNA was extracted and processed for TaqManRT-PCR analysis. Levels of EGF released into apical andbasolateral ALI compartments in response to CSE stimulationcompared with unstimulated controls were determined usinga commercially available DuoSet ELISA Development Kitfor EGF (R&D Systems) according to the manufacturer’s in-structions.

Analysis of EGF Receptor Expression and Activation. Western blotanalysis of the total EGF receptor (EGFR) protein expression incultured BCs and airway epithelium generated after 8 and 28 d ofthe ALI culture was performed following standard protocols (2)using rabbit monoclonal anti-EGFR antibody (clone D38B1,0.017 μg/mL; Cell Signaling Technology). Mouse monoclonalanti-GAPDH antibody (0.1 μg/mL; Santa Cruz Biotechnology)

was used as a loading control. To study the effect of EGF onEGFR phosphorylation, BCs were treated with 10 ng/mL EGFfor 5, 15, or 30 min or with medium only (Bronchial EpithelialBasal Medium; Lonza) and then processed for Western blotanalysis as described previously (2). Activated EGFR was de-tected using mouse monoclonal anti–phospho-EGFR antibody(Tyr-1173, clone 53A5, 0.29 μg/mL; Cell Signaling Technology),and total EGFR was analyzed as a control. Human phospho-receptor tyrosine kinase (RTK) antibody Proteome Profiler Ar-ray (R&D Systems) analysis was performed according to themanufacturer’s protocol.

Stimulation of Airway Epithelial Cells with EGF. The effect of EGFon BC morphology was evaluated at 48 h after direct stimulationof BCs in a submerged culture with 10 ng/mL EGF (Sigma-Aldrich) vs. unstimulated controls (Bronchial Epithelial BasalMedium only). The number of cells with fibroblast-like mor-phology (defined as elongation of the cytoplasm with formation offilopodia-like extensions with the total length ≥2 diameters of thenucleus of the same cell) was counted in three samples pergroup, each containing at least 200 cells.The effect of EGF on the capacity of BCs to generate dif-

ferentiated airway epithelium was analyzed using the ALI modeldescribed above. Beginning on day 0 of the ALI culture, EGF(10 ng/mL) was added every other day to the basolateral com-partment of the Transwell cultures to allow interaction with BCsduring the entire process of differentiation (28 d). Transepithelialresistance (Rt) was measured every other day when the media waschanged. At day 14, when the earliest morphological features ofairway epithelial differentiation are usually present (2), RNA wasisolated for analysis of the expression of genes related to airwayepithelial differentiation. At day 28, when BCs normally gener-ate differentiated airway epithelium (2), cells in different groupswere trypsinized, and cytospins were prepared for immunofluo-rescence analysis.To confirm that the effect of EGF on BCs is different from that

on differentiated cells, we applied EGF (10 or 20 ng/mL) toairway epithelia that had been fully differentiated in ALI culturefrom either apical (to expose differentiated cells) or basolateral(to expose BCs) sides every other day for 2 wk. In control groups,we applied ALI medium without added EGF. After stimulation,cell morphology was evaluated, Rt was measured, and RNA wasisolated as described above for TaqManRT-PCR gene expressionanalysis.

Analysis of Epithelial Barrier Integrity and Function. Epithelial bar-rier integrity of the ALI-derived airway epithelium was assessedby measuring Rt using a Millicell-ERS epithelial ohmmeter(Millipore). Epithelial barrier function was assessed by measuringparacellular permeability with an FITC-dextran flux assay. Inbrief, 1.5 mg/mL of FITC-dextran (40 kDa; Invitrogen) was addedto the apical surface of the airway epithelium differentiated inALI, and 100 μL of basal media were analyzed at different timepoints to measure the accumulation of fluorescence signal acrossthe epithelium using a SPECTRAmax GEMINI XS microplatespectrofluorometer (Molecular Devices) with an excitationwavelength of 485 nm and an emission wavelength of 538 nm.

Immunohistochemistry and Immunofluorescence Analysis. LAE sam-ples obtained during bronchoscopy from healthy nonsmokers andhealthy smokers, as described above, were analyzed by immuno-histochemistry using previously described protocols (1, 2) for the

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expression of EGF protein using mouse monoclonal anti-humanEGF antibody (1 μg/mL; R&D Systems), with a mouse IgG1(R&D Systems) isotype control. EGFR in the airway epitheliumwas localized by immunofluorescence analysis of biopsy sampleswith rabbit monoclonal anti-human EGFR antibody (1 μg/mL;Epitomics) and cytospin preparations of freshly isolated brushedLAE cells and BCs cultured from the LAE of healthy nonsmokerson chamber slides (Nunc) by immunocytochemistry and immu-nofluorescence analyses, respectively, using mouse monoclonalanti-human EGFR antibody (6.19 μg/mL; DAKO) or rabbitmonoclonal anti-human EGFR antibody (0.34 μg/mL; Cell Sig-naling; used only for BC samples as a confirmatory antibody).Cytospin preparations and histological sections of the airway

epithelium samples differentiated in ALI with and without EGFstimulation as described above were processed for immunoflu-orescence analysis (1). In brief, samples were blocked with 10%goat serum (Sigma-Aldrich) for 45 min and incubated with pri-mary antibodies mouse monoclonal anti-involucrin (IVL, 4 μg/mL;Thermo Scientific), rabbit polyclonal anti-KRT14 (3.6 μg/mL;Sigma-Aldrich), rabbit monoclonal anti-vimentin (VIM, 12μg/mL; Epitomics), and rabbit polyclonal anti-secretoglobin

1A1 (SCGB1A1, 20 μg/mL; BioVendor) for 3 h in a humidchamber at room temperature. Nuclei were counterstainedwith DAPI (Invitrogen). Rabbit polyclonal anti-KRT5 (4 μg/mL;Thermo Scientific) or mouse monoclonal anti-KRT5 (0.8 μg/mL;Thermo Scientific) antibodies were used for verification of the BCphenotype. Images were captured with a Zeiss Axiovert 200Mfluorescence microscope and analyzed with AxioVision Rel4.8 software. Quantitative analysis of cells expressing specificmarkers (EGF, EGFR, KRT5, IVL, and VIM) was performed asdescribed previously based on the immunohistochemistry (EGF)or immunofluorescence (EGFR, KRT5, IVL, and VIM; coex-pression with KRT5) analysis of the airway epithelial biopsysamples from healthy individuals (EGF; three samples, two im-ages per sample), cytopreparations of the airway epithelialbrushings from nonsmokers and smokers (EGFR, coexpressionwith KRT5; four samples per group, three images per sample),and cytopreparations and sections of the ALI-generated airwayepithelial cells derived from EGF-treated vs. control BCs (IVLand VIM, coexpression with KRT5; three samples per group,three images per sample). Representative images are provided inFigs. S2A, S3C, S8 A and B, and S9 A and B.

1. Shaykhiev R, et al. (2011) Cigarette smoking reprograms apical junctional complexmolecular architecture in the human airway epithelium in vivo. Cell Mol Life Sci 68(5):877–892.

2. Hackett NR, et al. (2011) The human airway epithelial basal cell transcriptome. PLoSONE 6(5):e18378.

A.

Nor

mal

ized

exp

ress

ion N.S.

p<0.05

Basal cells (days of culture)

EGFR ERBB4B.

Nor

mal

ized

exp

ress

ion

Basal cells (days of culture)

N.S.

p<0.05

0

1E-05

2E-05

3E-05

4E-05ERBB4

0 3 5 7 14 28 ALI (days of culture)

C.

Nor

mal

ized

exp

ress

ion

Control: p<0.01 (all time points vs. d0)

Fig. S1. Normalized expression of the EGFR (A) and v-erb-a erythroblastic leukemia viral oncogene homolog 4 (ERBB4) (B and C) genes during different timepoints of BC culture (n = 6) and ALI (n = 4). In A and B, normalized expression of the genes in the independent set of airway epithelium samples freshly isolatedby bronchoscopic brushings (in vivo; n = 6) is shown as well. Gene expression data are based on the TaqMan PCR analysis.

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KR

T5EG

FRM

erge

DAP

I

Set of samples # 1

A.

KR

T5EG

FRM

erge

DAP

I

Set of samples # 2

B.EGFR+ cells

C.

Basal cells (KRT5+)

EGFR+ (70.8 ± 9.3%)

EGFR-

KRT5+ (81.3 ± 6.4%)

KRT5-

EGFR KRT5 EGFR

Basal cells

DAKO Cell SignalingAnti-EGFR antibody

Fig. S2. Enrichment of EGFR expression in airway BCs. (A) Immunofluorescence colocalization of EGFR and KRT5 in the airway epithelial brushings obtainedfrom healthy individuals (n = 3, two images per sample). (B) (Left) Contribution (%) of KRT5+ cells to EGFR+ cells. (Right) Contribution (%) of EGFR+ cells toKRT5+ cells in the airway epithelial brushings obtained from healthy individuals based on the immunofluorescence analysis as shown in Fig. 1B and Fig. S2A. (C)Immunofluorescence analysis of cultured BCs for the expression of EGFR using different antibodies. (Left) Mouse monoclonal anti-EGFR antibody (DAKO).(Right) Rabbit monoclonal anti-EGFR antibody (Cell Signaling Technology), with cells costained for BC marker KRT5. (Scale bars: 20 μm.)

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Fold-change (log2) complete airway epithelium

smokers vs nonsmokers

p va

lue

(-log

)

EGFERBB4

TGFA

ERBB2

Up-regulated in smokers

Down-regulated in smokers

GPX20.71

CABYR0.70

PIR0.68

UCHL10.67

AKR1B100.71

ADH70.67

EGFAKR1C2

0.71

AKR1C10.69

AKR1C30.68

ME10.73

NQO10.67

A. B.

Non

smok

ers

Smok

ers

C.Sample 1 Sample 2 Sample 3 Sample 4

Sample 1 Sample 2 Sample 3 Sample 4

Fig. S3. Up-regulation of EGF in differentiated cells of the airway epithelium of smokers. (A) Volcano plot comparing expression of the v-erb-b homolog(ErbB) family of receptors and ligands in the complete LAE in healthy smokers (LAE-S; n = 31) and healthy nonsmokers (LAE-NS; n = 21). The y-axis showsBenjamini–Hochberg-corrected P values (−log); the x-axis, log 2-transformed fold change (LAE-S vs. LAE-NS). Red dots indicate significantly modulated genes (P <0.05, Benjamini–Hochberg correction). (B) Network of the oxidative stress-related genes with a high level of coexpression with the EGF gene in the airwayepithelium of smokers. The correlation was based on the genome-wide Pearson correlation analysis (r > 0.6; P < 0.05). Each circle represents an individual gene;the symbol and Pearson correlation coefficient r are indicated for each gene. Aldo-keto reductase family 1 (AKR1) members (orange circles) AKR1B10, AKR1C1,AKR1C2, and AKR1C3 are designated by orange circles. Other nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-dependent oxidative stress genes— alcoholdehydrogenase 7 (ADH7); ubiquitin carboxyl-terminal esterase L1 (UCHL1); pirin (PIR); calcium-binding tyrosine-(Y)-phosphorylation regulated (CABYR); gluta-thione peroxidase 2 (GPX2); malic enzyme 1, NADP+-dependent, cytosolic (ME1); and NAD(P)H dehydrogenase, quinone 1 (NQO1)—are designated by yellowcircles. (C) Representative images of the airway epithelium biopsy samples of nonsmokers and smokers (n = 4, three images per sample; for each phenotype, eachcolumn represents one sample) analyzed for EGF expression by immunohistochemistry. (Scale bars: 20 μm.)

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Basal cell phospho-RTK array

Con

trol

EGF

p-EGFR

PCPC

NC

Human phospho-RTK array coordinates

Fig. S4. Human phospho-RTK protein profiler array analysis of airway BCs stimulated for 15 min with EGF (10 ng/mL) vs. untreated control BCs showingphosphorylation of EGFR, but not of other RTKs, in EGF-treated BCs. (A list of all 49 RTKs included in the array is available at www.rndsystems.com/product_detail_objectname_RTKArray.aspx.)

Control

EGF

0

10

20

30

40

50

Control EGF

.B.Ap<0.02

% c

ells

with

elo

ngat

ed

fibro

blas

t-lik

e sh

ape

Fig. S5. Induction of a fibroblast-like morphology in airway BCs by EGF. (A) Representative micrographs of airway BCs after 48 h of stimulation with EGF(10 ng/mL) vs. unstimulated control BCs. (Scale bars: 20 μm.) (B) Comparison of the frequency of BCs with an elongated fibroblast-like shape (defined usingmorphological criteria, such as elongation of the cytoplasm along the anterior-posterior axis with formation of filopodia-like extensions in both directions withthe total length ≥2 diameters of the nucleus of the same cell) in EGF-treated vs. untreated control BCs (as in A; n = 3/group).

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0

0.0001

0.0002

0.0003

0.0004

0.0005

0

0.0004

0.0008

0.0012

0.000000

0.000003

0.000006

0.000009

0.000012

0.000015

0.000018

0

0.0001

0.0002

0.0003

0.0004

0.0005

0

4E-05

8E-05

0.00012

0.00016

Nor

mal

ized

exp

ress

ion

Control EGF

0 3 5 7 14 28

0 3 5 7 14 28 0 3 5 7 14 28

Days of ALI

0 3 5 7 14 28

0 3 5 7 14 28 0 3 5 7 14 28

Ciliated cell FOXJ1

Secretory (Clara) cell SCGB1A1

Serous secretory cell MUC5B

Tight junction CLDN3

Polarity complex PARD3

Mesenchymal (EMT-like) cell VIM

Control: p<0.005 (all time points vs. d0)

EGF vs. control: p<0.005 (d3-d28)

Control: p<0.005 (all time points vs. d0)

EGF vs. control: p<0.005 (d3-d28)

Control: p<0.005 (all time points vs. d0)

EGF vs. control: p<0.005 (d14, d28)

Control: p<0.05 (d3 vs. d0), p<0.005(d5-d28 vs. d0)

EGF vs. control: p<0.05 (d3), p<0.005(d5-d28)

Control: p<0.05 (d5 vs. d0, d14 vs. d0), p<0.005 (d28 vs. d0)

EGF vs. control: p<0.005 (d5, d14, d28), p<0.05 (d7)

Control: p>0.05 (all time points vs. d0)

EGF vs. control: p<0.005 (d3-d28)

Fig. S6. Airway epithelial differentiation from BCs in the ALI in the absence or presence of EGF treatment of BCs during ALI culture. Normalized expression ofgenes related to specific aspects of airway epithelial differentiation at different time points of ALI in the control and EGF-treated groups based on TaqMan PCRanalysis (n = 4 in each group) is shown. The significance of gene expression changes in the control group at different time points of ALI and between thecontrol and EGF-treated groups at specific time points of ALI is indicated for each gene.

A. B.

p<0.01

Basal side

Apical side

Rt(

Ohm

X c

m2 )

0

200

400

600

800

1000

1200

1400

1600

1 2 3 4 5EGF10 ng/ml

ControlControl EGF10 ng/ml

KRT5CD44KRT6AKRT6BKRT14IVLSFNSNAI2VIMFOXJ1DNAI1MUC5BSCGB1A1CDH1TJP1TJP3CLDN3OCLNPARD3PARD6B

all p>0.05

1 1.5 2-1.5-2

Fold-change EGF vs control

Fig. S7. Effect of the apical application of EGF on airway epithelial differentiation in the ALI culture. (A) Changes in expression of genes related to variousaspects of airway epithelial differentiation after apical application of EGF to the airway epithelium differentiated in ALI during the 2 wk-period vs. un-stimulated controls (n = 4 in each group). Complete gene names are provided in Figs. 3C and 4A. (B) Rt of the airway epithelium stimulated for 2 wk with EGF(10 ng/mL) from the apical (blue bars) or basolateral (yellow bars) sides of the ALI culture system compared with corresponding controls (n = 4 in each group).

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IVL(squamous cells)KRT5 (basal cells)

Control EGF

A.

VIM (mesenchymal cells)KRT5 (basal cells)

Control EGF

B.

Fig. S8. Modulation of airway epithelial differentiation by EGF. Immunofluorescence analysis of cytopreparations of the airway epithelium generated after28 d of ALI culture from BCs stimulated with EGF (10 ng/mL) vs. unstimulated controls for the expression of squamous cell marker IVL (A) and mesenchymal/EMTmarker VIM (B). Each sample was costained for the BC marker KRT5. Representative images for each group from two independent experiments are shown.(Scale bars: 20 μm.)

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IVL(squamous cells) KRT5 (basal cells)

Control EGF

A.

Control EGF

B.VIM (mesenchymal, EMT cells) KRT5 (basal cells)

Control EGF

C.DNAI1 (ciliated cells) KRT5 (basal cells)

Control EGF

D.SCGB1A1 (secretory cells) KRT5 (basal cells)

Fig. S9. Morphological changes in the airway epithelium generated from EGF-treated BCs. Immunofluorescence analysis of sections of the airway epitheliumsamples generated after 28 d of ALI culture from BCs stimulated with EGF (10 ng/mL) vs. unstimulated controls for the expression of squamous cell marker IVL(A); mesenchymal/EMT marker (B); ciliated cell marker dynein, axonemal, intermediate chain 1 (DNAI1) (C); and secretory cell marker SCGB1A1 (D). Each samplewas costained for the BC marker KRT5. (Scale bars: 20 μm.)

0

20

40

60

80

100

out of IVL+cells

out of VIM+cells

KR

T5+

cells

Fig. S10. Contribution (%) of KRT5+ (i.e., basal) cells to IVL-expressing and VIM-expressing cell populations in the airway epithelium samples generated after28 d of ALI culture from BCs stimulated with EGF (10 ng/mL) based on the immunofluorescence analysis described in Figs. S8 and S9.

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EGFR

EGF

Mucus-producing

(goblet) cell

Basal cell

Ciliated cell

Tight junctions

SCGB1A1-expressing

secretory cell

Altered generation of ciliated and SCGB1A1-expressing cells

Epithelial-mesenchymal transition-like

phenotype

Decreased barrier integrity

Squamous metaplasia

Normal airway epithelium

Smokers’ airway epithelium

Fig. S11. Diagram representing the mechanism of smoking-associated EGF-mediated alteration of airway epithelial differentiation described in the presentstudy. Cigarette smoke induces EGF expression in differentiated airway epithelial cells, mostly ciliated cells. EGF skews EGFR-expressing BCs toward abnormal(squamous and epithelial-mesenchymal transition-like) phenotypes, while altering BC differentiation into ciliated and SCGB1A1-expressing secretory cells andsuppressing formation of a stable airway epithelial junctional barrier.

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