TGF-� Induces Sustained Upregulation of SNAI1 andSNAI2 through Smad and Non-Smad Pathways in aHuman Corneal Epithelial Cell Line

Keiichi Aomatsu,1,2 Tokuzo Arao,2 Koji Sugioka,1 Kazuko Matsumoto,2 Daisuke Tamura,2

Kanae Kudo,2 Hiroyasu Kaneda,2 Kaoru Tanaka,2 Yoshihiko Fujita,2

Yoshikazu Shimomura,1 and Kazuto Nishio2

PURPOSE. The aim of this study was to investigate the expres-sion changes of epithelial mesenchymal transition (EMT)-re-lated molecules induced by TGF-� signaling in a human cornealepithelial cell line (HCECs).

METHODS. The cellular response to TGF-� was evaluated byimmunoblotting, quantitative real-time RT-PCR, and immuno-fluorescence microscopy in HCECs.

RESULTS. TGF-� significantly increased mRNA expression ofSNAI1, SNAI2, VIM, and FN1, but not TWIST1 through Smadand non-Smad pathways in HCECs. Protein expression of amesenchymal marker N-cadherin was dose-dependently in-creased and that of an epithelial marker of E-cadherin wasdecreased by TGF-�. TGF-�, but not EGF, mediated the EMT-like morphologic changes. Both TGF-� and EGF were capableof upregulating SNAI1 and SNAI2 by about two-fold within ashort response time. However, a detailed time course analysisrevealed drastically different expression patterns, with TGF-�mediating a sustained upregulation of SNAI1 and SNAI2 for atleast for 6 days and EGF allowing a return to the baselineexpression values after 8 � 12 h. These data indicate thatTGF-�, but not EGF, induces sustained upregulation of SNAI1and SNAI2 in HCECs.

CONCLUSIONS. TGF-� induces sustained upregulation of SNAI1and SNAI2 through Smad and non-Smad pathways, EMT-likemorphologic changes, downregulation of E-cadherin, and up-regulation of N-cadherin in HCECs. The authors’ findings pro-vide insight into the TGF-� signaling and the temporal expres-sion patterns of EMT-inducible transcription factors in HCECs.(Invest Ophthalmol Vis Sci. 2011;52:2437–2443) DOI:10.1167/iovs.10-5635

TGF-� is a multipotent growth factor that can exert multiplefunctions including the induction of cell proliferation, differ-

entiation, cell cycle arrest, apoptosis, and/or transformation in

time- and system-dependent manners by binding to transmem-brane serine/threonine kinase receptors.1,2 TGF-� is well-knownas a potent initiator of epithelial mesenchymal transition (EMT)and activates several transcription factors to induce EMT via Smador non-Smad pathways.1 These EMT-inducible transcription fac-tors are known as zinc finger factors (Snail, Slug, EF1, and SIP1)and basic helix-loop-helix factors (Twist, E2A, ID2/3, and E12/E47).1 Among the non-Smad signaling responses, activation ofextracellular signal-regulated kinases (ERKs), Rho GTPases, andthe PI3 kinase/AKT pathway in response to TGF-� have beenlinked to TGF-�–induced EMT through their regulation of distinctprocesses, such as cytoskeleton organization, cell growth, sur-vival, migration, or invasion.3 TGF-� also activates p38 MAP kinaseand induces EMT through p38 MAP kinase in NMuMG cells.4 Onthe other hand, EGF receptor (EGFR) activation enhances theEMT response in renal tubular epithelial cells,5 and EGFR coop-erates with integrin signaling to induce EMT via the upregulationof SNAI1 gene expression in cervical cancer cells.6 The EGF/EGFRsignaling pathways can also induce cancer cell EMT via STAT3-mediated TWIST gene expression.7

In the corneal epithelial cells, TGF-� enhances cellular migra-tion and inhibits cellular proliferation in corneal epithelial cells invitro and in vivo.8 During the wound healing of corneal epithe-lium, TGF-� is upregulated and the corneal epithelial cells migrateinto the injured area. The cells lack cellular proliferation in earlyphase, but begin to proliferate when epithelial defect is recov-ered.9 The corneal epithelial cells undergo phenotypic changes togain migratory characteristics in a way similar to the EMT processthrough activation of the p38 MAPK cascade during wound heal-ing.10 Regarding TGF-� receptors, TGF-� receptor-I and TGF-�receptor-II are both upregulated in cells migrating to cover acorneal wound after wounding.11 Collectively, TGF-� signaling isconsidered to play a critical role in corneal wound healing.

Notably, a recent report has demonstrated that the expressionlevels of Slug/SNAI2, a member of the Snail family of EMT regu-lator, was upregulated at sites of epithelial cell migration at themargins of normally healing corneal wounds, while did not occurat the margins of non-healing corneal erosions.12 Thus, the inves-tigation of the TGF-�-mediated phenotypes in corneal epithelialcells may be valuable to understand the corneal cell biology inwound healing. In this study, we investigated the expressionchanges of EMT-related molecules in human corneal epithelialcells (HCECs).

METHODS

Cell Cultures

The Sv40-immortalized human corneal epithelial cell (HCEC) line wasused in this study.13 HCECs were maintained in DMEM/F12 medium(Gibco, BRL, Grand Island, NY) supplemented with 15% fetal bovine

From the Departments of 1Ophthalmology and 2Genome Biology,Kinki University School of Medicine, Osaka, Japan.

Supported by funds for the Third-Term Comprehensive 10-YearStrategy for Cancer Control, the Program for the Promotion of Funda-mental Studies in Health Sciences of the National Institute of Biomed-ical Innovation (NiBio), and a Grant-in-Aid for Scientific Research (A).

Submitted for publication April 1, 2010; revised July 5, August 16,and September 6, 2010; accepted September 10, 2010.

Disclosure: K. Aomatsu, None; T. Arao, None; K. Sugioka,None; K. Matsumoto, None; D. Tamura, None; K. Kudo, None; H.Kaneda, None; K. Tanaka, None; Y. Fujita, None; Y. Shimomura,None; K. Nishio, None

Corresponding author: Kazuto Nishio, Department of GenomeBiology, Kinki University School of Medicine, 377-2 Ohno-higashi,Osaka-Sayama, Osaka 589-8511, Japan; knishio@med.kindai.ac.jp.

Biochemistry and Molecular Biology

Investigative Ophthalmology & Visual Science, April 2011, Vol. 52, No. 5Copyright 2011 The Association for Research in Vision and Ophthalmology, Inc. 2437

serum (FBS; Gibco) and gentamicin (40 �g/mL; Sigma, St. Louis, MO)at 37°C in a humidified incubator with 5% CO2. The A549 cell line wascultured in RPMI-1640 medium (Sigma) with 10% FBS.

Reagents

Human TGF-� and EGF were purchased from R&D Systems (Minneap-olis, MN). SB431542 (Sigma) and AG1478 (Biomol, Inc., St. Louis, MO),U0126 and Wortmannin (Cell Signaling Technology, Beverly, MA) weredissolved in dimethylsulfoxide for stock solution.

Western Blot Analysis

The antibodies used in this study were anti-Smad2, anti-phospho-Smad2, anti-EGFR, anti-phospho EGFR, anti-Akt, anti-phospho Akt, anti-MAPK, anti-phospho MAPK, anti-snail, anti-� actin, HRP-conjugatedsecondary antibody (Cell Signaling), anti-E cadherin, and anti-N cad-herin (Invitrogen, San Diego, CA). A western blot analysis was per-formed as described previously.14 The western blot experiments forevaluating signal transduction were performed under serum-starvedconditions (see Figs. 1A, 3A, 5A–D, 6D, and 6E), while the otherexperiments were performed in the presence of 15% serum because ofthe cellular damage that occurs as a result of serum-starved conditionsfor long periods of time. The densitometry analysis was performedusing commercial software (MultiGauge ver. 3.1 Fujifilm, Tokyo, Ja-pan). The experiment was performed in triplicate.

Cell Proliferation Assay

Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as described previ-ously.15 The experiment was performed in triplicate.

Real-Time Reverse Transcription–PCR

The real-time RT-PCR method was previously described.16 The follow-ing primers were used: SNAI1, forward 5�-TCT AGG CCC TGG CTGCTA CAA-3� and reverse 5�-ACA TCT GAG TGG GTC TGG AGG TG-3�;SNAI2, forward 5�-ATG CAT ATT CGG ACC CAC ACA TTA C-3� andreverse 5�-AGA TTT GAC CTG TCT GCA AAT GCT C-3�; TWIST1,forward 5�-GCC TTC TCG GTC TGG AGG AT-3� and reverse 5�-TTTCTC CTT CTC TGG AAA CAA TGA C-3�; vimentin, forward 5�-TGAGTA CCG GAG ACA GGT GCA G-3� and reverse 5�-TAG CAG CTT CAACGG CAA AGT TC-3�; fibronectin, forward 5�-GAG CTG CAC ATG TCTTGG GAA C-3� and reverse 5�-GGA GCA AAT GGC ACC GAG ATA-3�;and GAPD, forward 5�-GCA CCG TCA AGG CTG AGA AC-3� andreverse 5�-ATG GTG GTG AAG ACG CCA GT-3�. GAPD was used tonormalize the expression levels in the subsequent quantitative analy-ses.

Phase-Contrast Microscopy

The HCECs were cultured on 6-well plates. Morphologic changes weredetected and photographed using immunofluorescence microscopy(IX71-PAFM; Olympus, Tokyo, Japan) after stimulation with a growthfactor for 72 h. The percentage of spindle-shape cells was determinedfor each view.

Fluorescence Imaging

HCECs cultured on coverslips were fixed with 4% paraformaldehydefor 15 minutes at 37°C, then washed three times with PBS and blockedin 5% goat serum (DakoCytomation, Glostrup, Denmark) in PBS. Thecells were further incubated with primary antibody, anti-E cadherinand anti-N cadherin (1:500) (Invitrogen) for 1 hour, secondary AlexaFluor 488–conjugated goat anti-mouse IgG antibody (Invitrogen) for 1hour at room temperature. For the detection of F-actin rearrangement,the cells were fixed with 4% paraformaldehyde for 15 minutes at 37°C,then washed three times with PBS and permeabilized by a 5-minuteincubation with TBS solution (0.1% Triton X-100/1% BSA/PBS). Thecoverslips were again washed three times in PBS and immunostainedwith Rhodamine-conjugated Phalloidin (Invitrogen) for 15 minutes at

room temperature. The nuclei were subsequently stained for 5 minuteswith 4�,6-diamidino-2-phenylindole (DAPI). E-cadherin, N-cadherin,Rhodamine, and DAPI fluorescence were detected using immunofluo-rescence microscopy IX71-PAFM (Olympus) and E800 (Nikon, Tokyo,Japan).

Statistics

The statistical analyses were performed using commercial software(Excel; Microsoft, Redmond, WA) to calculate the average � SD and totest for statistically significant differences between the samples using aStudent’s t-test. A P-value � 0.05 was considered statistically signifi-cant.

RESULTS

TGF-� Upregulated SNAI1 and SNAI2 Expressionsin HCECs

TGF-� increased the phosphorylation levels of Smad2 in adose-dependent manner, and the effect was observed within15 minutes after stimulation (Fig. 1A). Generally, TGF-� is apotent growth inhibitor of epithelial cells in vitro. In line withthis feature, TGF-� significantly inhibited the cell proliferationof HCECs in a dose-dependent manner (Fig. 1B). This resultindicates that TGF-� signaling actually provides a signal re-sponse to HCECs.

Next, we examined the induction of EMT-inducible tran-scription factors by TGF-� stimuli. TGF-� upregulated themRNA expression of SNAI1 and SNAI2, which are key EMTregulators, but the expression of TWIST1 was not changed(Fig. 2A). The mRNA expression of vimentin and fibronectinwere upregulated (Fig. 2A).

FIGURE 1. TGF-� signaling actually provides a signal response to hu-man corneal epithelial cells (HCECs). (A) Western blot examining thephosphorylation and expression levels of Smad2 in time- (0, 15, 30, and60 min of TGF-� stimulation) and dose- (0.1, 1, and 10 ng/mL) depen-dent responses to TGF-� stimuli in HCECs. �-actin was used as aninternal control. Marker, protein size marker. (B) Growth inhibitoryeffect of TGF-� in HCECs evaluated using an MTT assay after incubationfor 0, 24, 48, and 72 h. The experiments were performed in triplicate.The error bars represent the SD. *P � 0.05.

2438 Aomatsu et al. IOVS, April 2011, Vol. 52, No. 5

Smad and Non-Smad Pathways Were Involved inTGF-�–Mediated Upregulation of SNAI1and SNAI2

Both Smad-mediated and non-Smad–mediated (ERK or AKT)pathways are involved in TGF-�–mediated EMT17; therefore,we examined the downstream signal pathway in HCECs. ThemRNA expression levels of SNAI1 and SNAI2 were upregulatedby TGF-� by about two-fold (Fig. 2B). Half of the level ofupregulation was effectively canceled by 10 �M of the MEKinhibitor U0126 or the phosphoinositide-3-kinase inhibitorWortmannin, and the upregulations of SNAI1 and SNAI2 werecompletely canceled by 10 �M of the TGF-� receptor inhibitorSB431542 (Fig. 2B). The expression of TWIST1 was apparentlyunchanged. In TGF-�–dependent responses, these data indi-cated that both Smad and non-Smad pathways were certainlyinvolved in TGF-�–mediated EMT in HCECs.

TGF-� Downregulated E-cadherin andUpregulated N-cadherin in HCECs

Western blotting revealed that the protein expression of amesenchymal marker N-cadherin was dose-dependently in-creased and epithelial marker of E-cadherin was slightly de-creased by TGF-� at 24�48 h after ligand stimulation in HCEC

(Fig. 3A). The expression level of E-cadherin was clearly de-creased and N-cadherin was increased in long-term treatmentof TGF-� at 4�6 days (Fig. 3B). In addition, HCECs expressedthe epithelium-specific marker keratin 12 at baseline, andTGF-� dose-dependently downregulated the expression of thismarker (Supplementary Fig. S1A, http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.10-5635/-/DCSupplemental). Westernblotting and immunocytochemistry revealed that TGF-� alsoupregulated the protein expressions of vimentin and fibronec-tin (Supplementary Fig. S1B, http://www.iovs.org/lookup/suppl/doi:10.1167/iovs.10-5635/-/DCSupplemental). Immuno-fluorescence analysis also showed that TGF-� decreased theexpression of E-cadherin on cellular membrane and increasedN-cadherin (Fig. 3C).

TGF-� Mediated EMT-like Morphologic Changesin HCECs

From a morphologic aspect, EMT is characterized by an in-crease in scattering and an elongation of the cell shape.18

TGF-� mediated both cell scattering and the elongation ofcell-shape in HCECs, whereas no effect was observed with EGF,which is also known to be an EMT-inducible ligand (Figs. 4A, 4B).Phalloidin/DAPI staining for the visualization of intracellular F-ac-

FIGURE 2. TGF-� upregulates EMT-related molecules via Smad and non-Smad pathways in HCECs. (A) ThemRNA expression levels of SNAI1, SNAI2, TWIST1, VIM (vimentin), and FN1 (fibronectin 1) weredetermined using a real-time RT-PCR analysis. HCECs were stimulated with TGF-� at 0.1, 1, and 10 ng/mLfor 24 h and the cells were used for analysis. (B) Real-time RT-PCR analysis for mRNA expression levels ofSNAI1 and SNAI2 via Smad or non-Smad signaling. HCECs were cultured with or without 10 �M of the MEKinhibitor U0126, 1 �M of the phosphoinositide-3-kinase inhibitor Wortmannin, or 10 �M of the TGF-� receptorinhibitor SB431542 for 30 min and were stimulated with 10 ng/mL of TGF-� for 8 h; the cells were thencollected for analysis. Rel mRNA, normalized mRNA expression levels (target gene/GAPD � 103). Theexperiments were performed in triplicate. The error bars represent the SD. *P � 0.05.

IOVS, April 2011, Vol. 52, No. 5 TGF-� Induces Sustained Upregulation of SNAI1 and SNAI2 2439

tin enabled the rearrangement of the cytoskeletal organizationfrom cell-cell borders into stress fibers in response to TGF-�,but not EGF, to be detected (Fig. 4A). The data showed that

TGF-�, but not EGF, mediates EMT-like morphologic changesin HCECs.

Inactive Crosstalk between TGF-� and EGFSignals in HCECs

Since crosstalk between the EGFR-ERK and TGF-�-Smad signal-ing pathways is known to enhance TGF-�–dependent re-sponses,19,20 we evaluated the crosstalk. The phosphorylationlevel of Smad2 increased in response to TGF-� stimulation andwas canceled by SB431542; however, EGF did not increase thelevel of phosphorylation (Fig. 5A). On the contrary, EGF mark-edly increased the phosphorylation levels of EGFR, ERK andAKT, while TGF-� slightly increased the p-ERK and p-AKTlevels (Fig. 5B). When both the TGF-� and EGF signals weresimultaneously activated, no increase in the phosphorylation ofSmad2 over EGF alone was noted (Fig. 5C). The p-ERK level,but not the p-EGFR or p-AKT levels, increased slightly with theaddition of TGF-� (Fig. 5D). These results indicate that there isno detectable crosstalk between TGF-� and EGF in HCECs.

Sustained SNAI1 and SNAI2 Upregulation Inducedby TGF-�, but Not EGF

TGF-�, but not EGF mediated the morphologic changes inHCECs (Figs. 4A. 4B); therefore we focused on the temporalregulation of SNAI1 and SNAI2 expression to explain thesedifferences. Notably, both TGF-� and also EGF significantlyupregulated the mRNA expressions of SNAI1 and SNAI2 byabout two-fold at 2 h after ligand stimulation (Fig. 6A). How-ever, a detailed time course analysis revealed that the upregu-lation of SNAI1 expression induced by EGF was immediatelyrestored to the baseline level within a short time period (�8 h)(Fig. 6B). Interestingly, TGF-� induced the sustained upregula-tion of SNAI1 and SNAI2 for �48 h (Fig. 6B), and furtheranalysis showed that the upregulation of SNAI1 and SNAI2 wassustained for at least 6 days (Fig. 6C). These findings indicatethat the temporal expression regulation of SNAI1 and SNAI2 byTGF-� or EGF occurs in very different manners. Western blot-ting showed that both TGF-� and EGF increased the short-termprotein expression of SNAI1 at 8 h; however, only TGF-�induced the sustained expression of SNAI1 at 24 h thereafter(Fig. 6C, 6D), indicating that the present data were consistentwith the results of real-time RT-PCR.

FIGURE 3. Protein expression regulation of N-cadherin and E-cadherinby TGF-�. (A) Western blotting for N-cadherin and E-cadherin wereexamined in HCECs and the EMT-positive control A549, a lung cancercell line. HCECs were stimulated with the indicated concentration ofTGF-� and the cells were collected at 24 h and 48 h. �-Actin was usedas an internal control. Marker, protein size marker. (B) Western blot-ting for N-cadherin and E-cadherin were examined in HCECs for longtime exposure. HCECs were stimulated with TGF-� at 10 ng/mL with-out serum starvation at indicated duration (4–6 days). �-actin was usedas an internal control. (C) Immunofluorescent staining of HCECstreated with TGF-� (10 ng/mL) or without (Cont.) for 6 days. HCECswere stained for N-cadherin (upper panels) and E-cadherin (lowerpanels) and the nuclei of cells were counterstained with DAPI. Scalebar, 25 �m.

FIGURE 4. TGF-�, but not EGF, me-diates EMT-like morphologic changesin HCECs. (A) Morphologic changeswere evaluated using phase-contrastmicroscopy (upper panels) and phal-loidin and DAPI staining under fluo-rescence microscopy (middle andlower panels). HCECs were culturedin the presence of 10 ng/mL of TGF-�or EGF for 72 h and then evaluated.Scale bar: (middle panels) 100 �m;(lower panels) 25 �m. (B) The ratioof spindle-shaped cells was then an-alyzed (ratio of cells with twofold �length/width in a high power field).Five random fields per sample at amagnification of �200 were cap-tured and evaluated. The error barsrepresent the SD. *P � 0.05.

2440 Aomatsu et al. IOVS, April 2011, Vol. 52, No. 5

DISCUSSION

TGF-� induced sustained upregulation of SNAI1 and SNAI2in HCECs. Lu et al.21 clearly demonstrated that EGF inducedEMT and the sustained upregulation of SNAI1 for up to 5days in A431 cell lines, and their data are consistent with thefindings of the present study. Although emerging data indi-cates that SNAI1 activity is regulated via post-transcriptionalregulatory mechanisms, such as its phosphorylation byGSK3—which leads to its inhibition by ubiquitination anddegradation,22 our results provide another regulatory mech-anism of SNAI1 expression at a transcriptional level. Mean-while, the upregulation of TWIST1 by TGF-� is known tooccur during the process of chondrocyte progression to-ward terminal maturation23 and during EMT via HMGA224 ina physiological context. In the corneal epithelial cells,TGF-� did not upregulate TWIST1 expression. This resultsuggests that TWIST1 is not likely to be induced by TGF-�signaling in HCECs.

Many studies have demonstrated crosstalk betweenTGF-�- and EGF-stimulated pathways through the facilitationof EMT. For example, the phosphorylation of the ERK-dependent R-Smad linker region enhances collagen I synthe-sis, implying positive crosstalk between the ERK and Smad

pathways in human mesangial cells.19 EGF plus TGF-� in-duced a dramatic morphologic change characteristic of EMTin rat intestinal epithelial (RIE) cells, and TGF-� augmentedthe EGF-mediated signaling of ERK and AKT by enhancingand prolonging the activation of the former and prolongingthe activation of the latter.20 EGF and TGF-� synergisticallystimulated proximal tubular cell migration after EMTthrough an increase in MMP-9 function and enhancedERK1/2 activation.25 Unexpectedly, the activation of theSmad pathway stimulated by the EGF signal was not seen inthis study, indicating that no detectable crosstalk existsbetween the TGF-� and EGF signal pathways in HCECs.

In general, EMT is a cellular process with a dramaticremodeling of the cytoskeleton and a losing of cell-cellcontacts.26 A hallmark of EMT is the loss of E-cadherinexpression and the cells undergoing EMT acquire expres-sion of mesenchymal components and manifest a migratoryphenotype.27 We found that TGF-� induced sustained up-regulation of SNAI1 and SNAI2 via both Smad and non-Smadpathways in HCECs (Fig. 7). Furthermore, TGF-� mediatedEMT-like morphologic changes, downregulated E-cadherin,and upregulated N-cadherin in HCECs. Although further invivo studies are necessary, these results suggest that TGF-�mediates the cellular phenotype toward EMT in HCECs.

FIGURE 5. Inactive crosstalk between TGF-� and EGF signals in HCECs. Western blot for phospho-Smad2 and Smad2 expression (A, C) andphospho-EGFR, -ERK, and -AKT and these protein expression levels (B, D) are shown. HCECs were cultured with or without exposure to SB431542or the EGFR tyrosine kinase inhibitor AG1478 for 30 min and stimulated with 10 ng/mL of TGF-� or EGF for 1 h or 15 min, respectively (A, B).For simultaneous stimulation with TGF-� and EGF, the HCECs were cultured with or without 10 �M of U0126, 1 �M of Wortmannin, or 1 �M ofAG1478 for 30 min and were then stimulated with 10 ng/mL of TGF-� and EGF for 1 h or 15 min, respectively (C, D). �-Actin was used as an internalcontrol. The experiments were performed in duplicate. Marker, protein size marker.

IOVS, April 2011, Vol. 52, No. 5 TGF-� Induces Sustained Upregulation of SNAI1 and SNAI2 2441

Taken together, our findings provide insight into the TGF-�signaling and the temporal expression patterns of EMT-inducible transcription factors in HCECs.

Acknowledgments

The authors thank Shinji Kurashimo and Yoshitaka Horiuchi for tech-nical assistance.

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FIGURE 6. TGF-�, but not EGF, induced the sustained upregulation of SNAI1 and SNAI2. (A) The mRNA expression levels of SNAI1 and SNAI2 weredetermined using a real-time RT-PCR analysis. HCECs were stimulated with 10 ng/mL of TGF-� or EGF for 2 h and the cells were analyzed. (B)Detailed time course analysis of the mRNA expression levels of SNAI1 and SNAI2 induced by TGF-� or EGF. HCECs were cultured in the presenceof 10 ng/mL of TGF-� or EGF for the indicated time course. Note that TGF-� resulted in the sustained upregulation of SNAI1 and SNAI2 for �48h. Rel mRNA, normalized mRNA expression levels (target gene/GAPD x 103). The error bars represent the SD. (C) Further time course analysis ofthe mRNA expression levels of SNAI1 and SNAI2 induced by TGF-�. HCECs were cultured in the presence of 10 ng/mL of TGF-� for the indicatedtime course. Normalized mRNA expression levels (target gene/GAPD x 103). The error bars represent the SD. *P � 0.05. (D) Western blotting forSNAI1/Snail1. HCECs were cultured in the presence of 10 ng/mL of TGF-� for the indicated time course and exposed to 10 �M of SB431542 at8 h. �-actin was used as an internal control. M, protein size marker. (E) Long-term expression of SNAI1 by TGF-� or EGF. HCECs were culturedin the presence of 10 ng/mL of TGF-� or EGF for the indicated time course. �-actin was used as an internal control.

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