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Inhibition of Epithelial–Mesenchymal Transition and Tissue Regeneration by Waterborne Titanium Dioxide Nanoparticles Xiaojiao Li, Lele Song, Xingjie Hu, Chang Liu, Jiye Shi, Hui Wang, Lixing Zhan, and Haiyun Song ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18986 • Publication Date (Web): 10 Jan 2018 Downloaded from http://pubs.acs.org on January 10, 2018

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ACS Applied Materials & Interfaces

Inhibition of Epithelial–Mesenchymal Transition and Tissue Regeneration by Waterborne Titanium Dioxide Nanoparticles Xiaojiao Li1,5, Lele Song1,5, Xingjie Hu2, Chang Liu1, Jiye Shi3, Hui Wang4,*, Lixing Zhan1,*, Haiyun Song1,4,* 1

Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China

2

School of Public Health, Guangzhou Medical University, Guangdong 511436, China

3

UCB Pharma, Slough, SL2 3WE, United Kindom

4

School of Public Health, Shanghai Jiao Tong University, Shanghai 200025, China

5

These authors contributed equally to this work.

*Emails: [email protected]; [email protected]; [email protected] KEYWORDS:

titanium

dioxide

nanoparticles,

transition, TGFβ signaling, colitis, tissue regeneration

ACS Paragon Plus Environment

epithelial-mesenchymal

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Titanium dioxide nanoparticles (TiO2NPs) are among the most widely manufactured nanomaterials with broad applications in food industry, cosmetics and medicine. Whereas the toxicity of TiO2NPs at high doses has been extensively explored, potential health risks of TiO2NPs exposure at non-toxic concentrations remain poorly understood. Epithelial-mesenchymal transition (EMT) plays pivotal roles in a diversity of physiological and pathological processes including tissue regeneration and cancer metastasis. In this study, we find that cellular uptake of TiO2NPs inhibits EMT-mediated cell remodeling and cell migration without exhibiting cytotoxicity. Further investigation reveals that TiO2NPs suppress the process of EMT through the blockade of TGFβ signaling. Particularly, TiO2NPs interact with the TGFβ receptor TβRI/II complex, induce its lysosomal degradation, and thereby down-regulate expression of TGFβ target genes. Moreover, we show that waterborne TiO2NPs do not elicit toxicity in healthy tissues, but hamper EMT-mediated wound healing in two animal models. Long-term exposure of TiO2NPs in environmental water and drinking water impede regeneration of amputated fin in zebrafish and recovery of intestinal mucosal damage in colitic mice, respectively. Our results reveal previously unknown effects of TiO2NPs during tissue remodeling and repair, which have significant implications in their risk assessment and management.


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INTRODUCTION

Nanotechnology develops rapidly in biological and medical research as well as in modern industries. Nanoparticles (NPs) exhibit great potentials in sensing,1-4 targeting,5-8 delivering,9-13 imaging biomolecules and biological structures.14-20 Recently, various studies have highlighted the effects of NPs on perturbing cellular activities and functions, which can initiate a wide range of biological responses. However, the conclusions are controversial. Whether NPs can induce toxicity in cell and animal models largely relies on not only their physicochemical properties, but also their doses, duration and patterns of exposure.21-23 On the other hand, it is also important to explore the toxicity-independent effects of NPs in various biological processes to fully understand their health risks or benefits. As examples, magnetic iron oxide NPs demonstrate a catalase-like activity and positively affect aging and neurodegeneration in Drosophila;24 silica NPs and nanodiamonds induce degradation of Wnt signaling components, thereby interfering with a diversity of processes including embryonic development and lipid metabolism.25-26

Nanoscale titanium dioxide (TiO2) is among the most widely manufactured NPs being applied to medicine, cosmetics and food industries. TiO2NPs are used as transducer materials for microfluidic biosensors to obtain enhanced catalytic activity and improved stability and sensitivity.27-28 As an example of medical



application, such microfluidic devices have been used for the detection of biomarkers of breast cancer.29 TiO2NPs are also capable of delivering a wide range of chemical compounds, antibodies or functional nucleic acids for cancer therapy.30-31 In addition, TiO2NPs are extensively used as ingredients of food additives, cosmetics and sunscreens.32 Therefore, the potential health risks of TiO2NPs in living systems, especially during long-term exposure, attract broad attention. A study focusing on the cosmetic use of TiO2NPs suggests that TiO2NPs exposure can induce autophagy in human keratinocytes,33 whereas another study focusing on foodborne TiO2NPs finds that TiO2NPs only elicit minimum biological responses in cultured intestinal cells.34 Nevertheless, the long-term effects of foodborne TiO2NPs at non-toxic concentrations are largely unexplored. It is necessary to investigate potential health risks of orally administrated TiO2NPs on digestive organs in vivo under both physiologic and pathological contexts.

Epithelial-mesenchymal transition (EMT) refers to the change of cell identity from the epithelial to the mesenchymal phenotype, during which extensive remodeling of cellular structure occurs to promote cell migration. EMT plays critical roles in a variety of biological contexts, including embryonic development, cancer metastasis, and wound healing and tissue regeneration.35-37 The process of EMT is regulated by many extracellular signals, among which the transforming growth


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factor–β (TGFβ) signaling pathway plays a central role.38-40 Particularly, TGFβ signaling-mediated EMT is indispensable for tissue regeneration and wound repair.41-42 The TGFβ ligand has a type I receptor (TβRI) and a type II receptor (TβRII) localized on the cell membrane.43-44 TGFβ directly binds to TβRII but not TβRI. However, this interaction induces the phosphorylation and activation of TβRI by TβRII. The activated TβRI then recruits the Smad family transcription factors (Smad2 and Smad3) for phosphorylation, which in turn enter the nucleus to modulate gene transcription.45-46

In this study, we explored biological consequences of TiO2NPs exposure within the doses that did not elicit toxicity in cells and tissues. We found that pre-incubation with TiO2NPs prevented TGFβ-induced acquiring of mesenchymal phenotypes in epithelial cells. Consistently, TiO2NPs treatment also attenuated the pro-migratory effect of TGFβ. On the molecular level, we demonstrated that TiO2NPs interacted with the TβRI/II receptor complex. Consequently, the internalization of TiO2NPs from the cell surface delivered TGFβ receptors to the lysosomes for degradation, and thereby inhibited Smad2/3 activation and downstream gene expression. We further showed that inhibition of TGFβ signaling and EMT by TiO2NPs had a negative effect on wound healing and tissue regeneration in vivo. TiO2NPs exposure via environmental water impaired regeneration of amputated caudal fin in zebrafish. Whereas long-term exposure of



TiO2NPs via drinking water did not cause intestinal mucosal damage, it significantly hampered the recovery of colitis in mice. Our work thus revealed potential health risks of TiO2NPs exposure under pathological contexts and disclosed the mechanism underlying these effects.

RESULTS AND DISCUSSION

Physiochemical Properties and Cytocompatibility of TiO2NPs

The TiO2NPs used in our study had a primary size of 50 nm and an apparent hydrodynamic size of 124 nm, as determined by transmission electron microscopy (TEM) and dynamic light scattering (DLS) analyses (Figure S1). The zeta potential of these NPs was about -27 mV (Figure S1b). We evaluated the biocompatibility of TiO2NPs in A549 and HEK293, two types of well-characterized epithelial cell lines. At selected concentrations between 10 and 200 µg/mL, incubation with TiO2NPs for 24 hours did not compromise the viability of A549 cells (Figure S2a). We also monitored the cell viability at different time points after incubation with 100 µg/mL TiO2NPs, and no cytotoxicity was detected within 48 hours (Figure S2b). Similar results were observed in TiO2NPs-treated HEK293 cells, suggesting a good cytocompatibility of TiO2NPs in these cells (Figure S3a,b).

TiO2NPs Suppress TGFβ-Induced Epithelial-Mesenchymal Transition


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Although incubation with a wide range of TiO2NPs concentrations did not compromise the viability of A549 or HEK293 cells, we continued to investigate whether cellular uptake of TiO2NPs could affect certain cell functions in the absence of apparent cytotoxicity. The process of EMT requires structural reprogramming of epithelial cells into mesenchymal cells, such as the loss of cell polarity and cell–cell adhesion, and the acquirement of migratory and invasive properties. TGFβ signaling plays a pivotal role in EMT, decreasing the level of cell adhesion protein E-cadherin while increasing that of the mesenchymal marker N-cadherin (Figure 1a).38, 47 As this transition is accompanied with morphological changes in the cell shape, we could easily examine the effect of TiO2NPs on this important cellular activity by optical microscope.48 As expected, the addition of TGFβ ligand in cultured A549 cells induced morphological changes from a pebble-like shape to a spindle shape, a characteristic of EMT (Figure 1b). Pre-incubation with 100 µg/mL TiO2NPs noticeably reduced the morphological changes elicited by TGFβ, whereas it did not exert any effect on the shape of A549 cells in the absence of TGFβ ligand (Figure 1b). At the molecular level, TGFβ significantly induced downregulation and upregulation in the protein levels of E-cadherin and N-cadherin, respectively, while TiO2NPs exposure largely restored the protein levels of these cell markers (Figure 1c,d). Consistent with its effect on the cell morphology, treatment with TiO2NPs alone did not alter the levels of E-cadherin or N-cadherin (Figure 1c,d). These results imply that



TGFβ-induced EMT can be weakened by TiO2NPs in A549 cells.

TiO2NPs Exposure Inhibits TGFβ-Mediated Cell Migration

EMT has a crucial role in tissue regeneration and wound healing, during which the activation of TGFβ signaling promotes cell migration.36 We explored the biological consequences of EMT-inhibition by TiO2NPs in two cell-based migration assays, namely the wound healing assay and the transwell assay. In the wound healing assay, a scratch made on confluent epithelial cells can gradually close up via cell migration (Figure 2a). TGFβ stimulation markedly increased the migratory speed of A549 cells, whereas pre-incubation with 100 µg/mL TiO2NPs diminished this pro-migratory effect of TGFβ (Figure 2b,c). In the transwell assay, cells plated in the upper chamber can move into the lower chamber depending on their migratory ability (Figure 3a). The addition of TGFβ in the cell medium significantly increased the numbers of A549 cells migrating into the lower chamber in the absence of TiO2NPs. However, this effect was also weakened by pre-incubation with 100 µg/mL TiO2NPs (Figure 3b,c). Notably, incubation with TiO2NPs alone did not hamper cell migration in above two assays, suggesting that the observed effects of TiO2NPs on cell migration were caused by inhibition of TGFβ signaling rather than general cell toxicity.

TiO2NPs Attenuate Transcriptional Outputs of TGFβ Signaling


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Based on the observation that cellular uptake of TiO2NPs hampered TGFβ-induced onset of EMT and subsequent cell migration, we asked whether intracellular TiO2NPs affected the transcriptional outputs of TGFβ signaling. Therefore, we collected the A549 cells after the wound healing assay and analyzed the mRNA levels of TGFβ target genes essential for EMT and cell migration, including HMGA2, Slug and Zeb-1.39,

49-50

In accordance with the

results of wound healing and transwell assays, the addition of TGFβ in the cell medium upregulated the expression of these genes in A549 cells. This effect of TGFβ was strongly dampened by pre-incubation with 100 µg/mL TiO2NPs, although equal dose of TiO2NPs had no effect on the expression of these genes in the absence of TGFβ (Figure S4a).

TGFβ signaling can regulate target gene expression via activating the transcription factor Smad family proteins, or via cross-talking with other signaling pathways.46 With a synthetic reporter construct that specifically responds to activated Smad2/3,51 we determined whether these NPs inhibited the Smad-dependent, canonical TGFβ signaling. The reporter gene had only basal expression in the absence of TGFβ, and TGFβ stimulation resulted in over ten-fold induction in its expression levels. Treatment with 10-100 µg/mL TiO2NPs did not alter basal expression of the reporter in A549 cells. In contrast, they attenuated TGFβ-stimulated expression of the reporter in a dose dependent



manner (Figure S4b). Next, we measured expression levels of the reporter at various time points after TiO2NPs exposure. Pre-treatment with 100 µg/mL TiO2NPs exhibited continuous inhibition on TGFβ-induced reporter expression without detectable effects in the basal condition (Figure S4c). These results confirmed that TiO2NPs weakened Smad-mediated transcription of TGFβ target genes, which in turn affected EMT and cell migration.

TiO2NPs Promote Lysosomal Degradation of TGFβ Receptors

Having found that intracellular TiO2NPs could diminish Smad-dependent transcriptional outputs of TGFβ signaling, we further investigated the underlying molecular mechanism. The protein levels of Smad2 and Smad3, two major Smad proteins activated by TGFβ signal,45 were not affected by TiO2NPs incubation in A549 cells. In contrast, the activities of both Smad2 and Smad3, marked by the levels of Smad phosphorylation at specific C-terminal serine residues,52-53 were largely reduced in the presence of TiO2NPs (Figure 4a). Transduction of TGFβ signal to Smad2/3 requires binding of TGFβ ligand to the TβRII-TβRI receptor complex, which in turn induces phosphorylation events on Smad2/3.45-46 We therefore examined whether cellular uptake of TiO2NPs could affect these membrane receptors. In support of this notion, western blotting analysis displayed obvious reduction in the protein levels of TβRI and TβRII after A549 cells were incubated with TiO2NPs (Figure 4a). This phenomenon did not reflect a general


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effect on cellular proteins, since TiO2NPs did not affect the protein levels of Smad2 or Smad3. Neither did they alter the levels of Lrp6,54 another type of receptor localized on the cell membrane (Figure 4a). In addition, we also observed similar effects in HEK293 cells, suggesting that the downregulation of TGFβ receptors by TiO2NPs exposure could occur in various types of epithelial cells (Figure S5).

After TGFβ binds to its receptors, they invaginate together from the cell surface to form the early endosome, where the ligand is targeted for the lysosome and most of the receptors are recycled back to the cell membrane for the next round of signaling activation.55 It has been reported that endocytosed TiO2NPs are also delivered to the lysosome.56 We thus investigated whether TiO2NPs could interfere with the trafficking pathway of TGFβ receptors. In the absence of TiO2NPs, less than 20% TβRI and TβRII colocalized with the lysosome marker Lamp1, indicating that the routine turnover of TGFβ receptors occurred in this organelle. Incubation with 100 µg/mL TiO2NPs induced more than 70% TβRI and TβRII localizing in the lysosome within 24 hours (Figure 4b-d). These results confirmed that TiO2NPs downregulated the protein levels of TGFβ receptors via promoting their lysosomal transport. Next, we examined possible interaction between TiO2NPs and TGFβ receptors. After incubation with the lysate of A549 cells, we analyzed proteins bound on TiO2NPs. Both TβRI and TβRII were



pulled-down by TiO2NPs, whereas Smad2, Smad3 or Lrp6 did not bind to TiO2NPs (Figure 4e). Therefore, TiO2NPs may serve as nanocarriers to deliver the TβRII-TβRI receptor complex to the lysosome.

TiO2NPs Impair Caudal Fin Regeneration in Zebrafish

Next, we explored the biological consequences of EMT-inhibition by TiO2NPs with in vivo models. TGFβ signal-mediated EMT plays an evolutionally conserved role in tissue regeneration. The zebrafish fin possesses a relatively simple structure with limited cell types. Amputation of caudal fins is easily operated and does not compromise the viability, thereby providing a valuable model to study tissue regeneration (Figure 5a).42 The regeneration of amputated caudal fins could be completed within 7 days in the absence of TiO2NPs. However, nurturing zebrafish in water containing 100 µg/mL TiO2NPs significantly retarded the speed of recovery (Figure 5b and Figure S6). It was reported that TGFβ singling was activated 24-72 hours after fin amputation, which was essential for fin regeneration. We monitored the transcriptional activity of TGFβ singling in the caudal fin during this period. In control fins, amputation induced expression of Integrin-β5,42 a TGFβ singling target gene required for EMT-mediated wound healing. This effect was almost abolished upon water-mediated TiO2NPs exposure, indicating that TiO2NPs impaired the regeneration of caudal fin by attenuating the activity of TGFβ signaling (Figure 5c).


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Dietary TiO2NPs Hamper the Recovery of DSS-Induced Colitis by Weakening TGFβ Signaling Given TiO2NPs are extensively used in food industry as a type of food additives,34 we investigated the long-term effects of dietary TiO2NPs, administrated via drinking water, on intestinal homeostasis under both physiological and pathological conditions. We first examined the effects of dietary TiO2NPs in colons of normal mice. Age- and sex- matched C57BL/6 mice had been fed with normal drinking water or water containing 100 µg/mL TiO2NPs for 4 weeks before colon tissues were analyzed (Figure 6a). Dietary TiO2NPs did not affect the colon length, an important parameter for intestinal health (Figure 6b,c). Notably, the H&E staining of colon tissues did not reveal any noticeable difference between the colons from control mice and those from mice fed with TiO2NPs (Figure 6d). These results suggest that dietary TiO2NPs of reasonable doses may not cause harmful effects on the colon under physiological context. Dietary dextran sodium sulphate (DSS) can induce colitis in mice,57 which can gradually

recover

after

the

administration

of

DSS

is

halted.58

This

well-characterized model is widely used for the study of intestinal regeneration. It has been reported that TGFβ signaling-mediated EMT is required for the recovery of DSS-induced intestinal mucosal damage.59-60 To investigate whether dietary TiO2NPs affect TGFβ-regulated recovery of colitis, mice were exposed to



TiO2NPs via drinking water for 4 weeks, with DSS induction throughout the third week (Figure 7a). The control mice and TiO2NPs-treated mice showed similar values in the body weight and the disease activity index (DAI) until the completion of DSS induction, indicating the same extent of pathological damages. During the recovery period, TiO2NPs-treated mice displayed severe weight loss and increased DAI comparing to the control mice (Figure 7b,c). We further analyzed the length and histology of the colons. On the day 21, the ending point of DSS induction, control mice and TiO2NPs-treated mice displayed similar colon lengths, mucosal damage and histology scores (Figure 7d-g), indicating that TiO2NPs did not strengthen DSS-induced colitis. However, TiO2NPs-treated mice possessed shorter colons, more severe intestinal inflammation and higher histology scores during recovery (Figure 7d-g). These data suggest that long-term uptake of TiO2NPs may hinder the repair of colon injury. Lastly, we investigated the effects of dietary TiO2NPs on TGFβ signaling after colon injury. Consistent with our observations in cultured cells, dietary TiO2NPs also dramatically decreased the protein levels of TβRI and TβRII, and the activity of Smad2 in colon tissues (Figure 7h). Consequently, expression of TGFβ target genes required for intestinal regeneration was impaired in colons from TiO2NPs-treated mice (Figure 7i). These results suggested that dietary TiO2NPs impeded the recovery of DSS-induced colitis via inhibiting TGFβ signaling.


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CONCLUSION

In summary, our study identifies a cytotoxicity-independent effect of TiO2NPs on an important cellular function, namely epithelial-mesenchymal transition. With cell-based assays, we find that cellular uptake of TiO2NPs blocks the morphological changes and migratory activities of epithelial cells acquired in the process of EMT. We show that TiO2NPs inhibit EMT via their capacity to weaken TGFβ-triggered signal transduction and target gene expression. Specifically, TiO2NPs bind to TGFβ receptors TβRI/II, disturb their endocytic trafficking and recycling, and deliver them to the lysosomes. In addition, we observe that environmental exposure of TiO2NPs hinders caudal fin regeneration in zebrafish, and dietary exposure of TiO2NPs via drinking water hampers intestinal repair of colitic mice. These results indicate that inhibition of TGFβ signaling and EMT by TiO2NPs may bring significant impacts on pathological conditions such as wound healing and digestive diseases. Given the extensive use of TiO2NPs as ingredients of food additives and cosmetics, our study discloses previously unreported health risks for long-term exposure of TiO2NPs.

METHODS AND EXPERIMENTAL SECTION

Cell Culture

Human lung epithelial A549 cells and human embryonic kidney HEK293 cells



were obtained from the Cell Bank of Chinese Academy of Sciences, and cultured at 37°C in a humidified incubator with 5% CO2. RPMI 1640 medium, DMEM medium, fetal bovine serum (FBS), streptomycin and penicillin were from Thermo Fisher Scientific.

Characterization and Cytotoxicity of TiO2NPs

TiO2NPs were from DKNano Technology. To measure their primary particle size, TiO2NPs solution was dropped onto carbon coated copper grids to evaporate excessive solvent, and examined with transmission electron microscopy (Jeol 2010, 200 KV). The apparent hydrodynamic size and the zeta potential of TiO2NPs in cell culture medium were measured with a Delsa Nano C analyzer (Beckman Coulter). The cytotoxicity of TiO2NPs in A549 and HEK293 cells was measured withthe Cell Counting Kit-8 (CCK-8, Sigma-Aldrich). Briefly, cells were cultured in 96-well plates at a density of 5000 cells/well, and incubated with TiO2NPs of various concentrations for indicated time. After wash with phosphate buffer saline (PBS), CCK-8 solution was added to cells and incubated for 2 hours at 37°C. The absorbance was measured at 450 nm using a microplate reader (Bio-Rad 680).

Wound-Healing Assay

A549 cells were cultured in 12-well plates. Confluent cells were incubated with


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100 µg/mL TiO2NPs for 3 hours, scratched with 200 µL sterile pipet tips, and washed three times with PBS. Cells were then cultured in RPMI 1640 medium with 2% FBS in the absence or presence of 5 ng/mL TGFβ (R&D systems).Phase contrast microscopy images were recorded at 0 hour and 36 hour after scratching. These cells were then used for measurement of TGFβ target gene expression. The expression of actin gene was used as an internal control. The migration rates under various conditions were quantified with the ImageJ software.

Transwell Assay

A549 cells were incubated with 100 µg/mL TiO2NPs for 3 hours, washed 3 times with PBS, re-suspended in 200 µL of serum free RPMI 1640 medium and seeded in the upper well of the Costar Transwell System (Corning). The bottom wells were filled with 700 µL RPMI 1640 medium with 10% FBS in the absence or presence of 5 ng/mL TGFβ. After incubation for 36 hours, non-migrated cells were removed from the upper surface of the chamber with a cotton swab. Cells that migrated into the lower chamber were fixed with 4% paraformaldehyde (PFA), washed with PBS and stained with 0.1% crystal violet. Phase contrast microscopy images were recorded and quantification of migrated cells was performed with the ImageJ software.

Real-Time PCR



Total RNAs from A549 cells after the wound healing assay were extracted with TriZol (ambion) and reverse-transcribed into complementary DNAs with the ReverTraAce qPCR RT Kit (TOYOBO). Quantitative PCR was performed with SYBR Green Realtime PCR Master Mix (TOYOBO) in the StepOne Real-Time PCR System (Applied Biosystems). The expression level of actin gene was used as an internal control.

Dual-Luciferase Assay

The p(CAGA)12-MLP-Luc reporter and pRL-TK renilla luciferase reporter were co-transfected in HEK293 cells with the Lipofectamine 3000 reagent (Thermo Fisher Scientific). One day after transfection, cells were incubated with TiO2NPs of various concentrations (10, 50, 100 µg/mL) for 3 hours and washed 3 times with PBS. TGFβ1 induction (at 100 pM) was performed for 24 hours or indicated time. Cells were then lysed and luciferase activities were measured with the Luciferase Assay System (Promega). All Firefly luciferase activities were normalized to Renilla luciferase activities.

Western Blotting and Confocal Imaging

Whole cell lysate was analyzed by western blotting after cells were incubated with 100 µg/mL TiO2NPs for 3 hours and cultured for additional 16 hours. The following antibodies were used. Rabbit anti-TβRI (1:1000 for western blot and 1:200 for


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immunostaining), rabbit anti-TβRII (1:1000 for western blot and 1:200 for immunostaining) were from Santa Cruz Biotechnology. Rabbit anti-Smad2 (1:1000), rabbit anti-Smad3 (1:1000), rabbit anti-p-Smad2 (1:1000), rabbit anti-p-Smad3 (1:1000), rabbit anti-Hsp90 (1:1000) were purchased from Cell Signaling Technology. Mouse anti-Tubulin (1:10000), goat anti-rabbit IgG-HRP (1:3000), goat anti-mouse IgG-HRP (1:3000) were from Abcam. Goat anti-rabbit Alexa Fluor488 (1:500) was from Thermo Fisher Scientific. Rabbit anti-Lrp6 (1:1000) was generated by Abclone Technology.

To investigate the subcellular localizations of TGFβ receptors, A549 cells were transfected with RFP-tagged Lamp1 for the labeling of the lysosomes. On the next day, cells were incubated with 100 µg/mL TiO2NPs for 3 hours and cultured for additional 16 hours. Cells were fixed in 4% PFA for 20 minutes at room temperature, washed three times in PBS containing 0.1% TritonX-100 (PBST) and blocked with PBST containing 5% bovine serum albumin for one hour. Cells were incubated with the primary antibody against TβR1 or TβRII at 4°C overnight and the goat anti-rabbit Alexa Fluor488 at room temperature for 2 hours, and followed by PBST washing, 4′,6-diamidino-2-phenylindole (DAPI) staining and fluorescence imaging with a confocal laser scanning microscope (510 NLO, Zeiss). The co-localizations of TβRI or TβRII (green) with Lamp1 (red) were quantified with the ImageJ software. Two fluorescent signals were measured and



tMr values (thresholded Mander’s coefficients) indicating the percentage of green signals colocalized with red signals in merged images were calculated. The values represented mean ±SD on the basis of analysis of 20 randomly selected cells. For depiction of the fluorescence intensity profile, the trajectories of red and green signals were built by pairing spots in each frame using the single-particle tracking plugin of ImageJ.

Zebrafish Caudal Fin Regeneration Assay

The wild type zebrafish AB strain was maintained at 28.5 °C in an Aquatic Habitats for Accelerated Bioresearch (AHAB) recirculating filtered water system (Aquatic Habitats) on a 14-hour/10-hour light/dark cycle. All experiments were performed using 18-month-old, male zebrafish. For caudal fin amputations, fish was anesthetized in 0.1% tricaine and fins were cut with razor blades. After amputation, fish was cultured in water in the absence or presence of 100 µg/mL TiO2NPs. Images of caudal fins were acquired with a Motic SMZ168 Stereo Zoom microscope. 24 hours post-amputation, caudal fins were cut and homogenized in 0.5 mL Trizol reagent. Real-time PCR was performed with the expression of actin as an internal control.

The effect of Dietary TiO2NPs on the Recovery of DSS-Induced Colitis

7-week-old, male C57BL/6 mice were fed with normal drinking water or water


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containing 100 µg/mL TiO2NPs for 28 days. From the day 15, 3% DSS (w/v) (36-50 kDa; MP Biomedicals) was added in drinking water for 7 days. Body weight, diarrhea and occult blood in feces were recorded daily to evaluate the clinical disease activity index (DAI). DAI is based on assigning a scoring system of 0-4 for each parameter. Score 0: no weight loss, normal stool and no blood; score 1: 1–3% weight loss; score 2: 3–6% weight loss, loose stool, blood visible in stool; score 3:6–9% weight loss; score 4: >9% weight loss, diarrhea with gross bleeding. The lengths of colon were measured after the mice were sacrificed. All mouse and fish experiments were reviewed and approved by the Animal Care and Use Committee of Shanghai Institute of Nutritional Sciences, Chinese Academy of Sciences.

Colon Histology

After the mice were sacrificed, an approximately 1 cm length of the distal colon was collected, fixed in 4% PFA, dehydrated, embedded in paraffin, and sectioned using standard methodologies. Colons were examined using 5 µm thick, 200 µm deep sections stained with haematoxylin and eosin (H&E) and evaluated by light microscopy for histology scores. For tissue damage: score of 0, no mucosal damage; score of 1, mucosal lesions; score of 2, surface mucosal erosion or focal ulceration; score of 3, extensive mucosal damage and extension into deeper structures of the bowel wall. For inflammatory cells infiltration: none inflammatory



cells infiltration was scored as 0; slightly dispersed cells infiltration was scored as 1; moderately increased cell infiltrates forming occasional cell foci was scored as 2; severely large area of cell infiltrates causing loss of tissue architecture was scored as 3. For crypt injury: none, 0; basal 1/3 damaged, 1; basal 2/3 damaged, 2; loss of surface epithelium, 3; loss of entire crypt and epithelium, 4. The histology scores are combined scores from that of tissue damage, inflammatory cells infiltration and crypt injury.

AUTHOR CONTRIBUTIONS

H.S., L.Z. and H.W. designed experiments, H.S. and X.L. wrote the manuscript. X.L., C.L., X.H. and J.S. performed cell-based experiments and analyzed the results. X.L., L.S. and X.H. performed mouse-based experiments and analyzed the results.

ACKNOWLEDGEMENTS

This study was supported by the National Natural Science Foundation of China (81773434 and 31571498), the Key Research Program of the Chinese Academy of Sciences (KFZD-SW-213), the National Program on Key Research Project of China (2017YFC1600104) and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA12010102).

COMPETING FINANCIAL INTERESTS


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The authors declare no competing financial interests.

SUPPORTING INFORMATION

Figures S1−S6: the physicochemical properties of TiO2NPs; the cytotoxicity of TiO2NPs in A549 cells; the cytotoxicity of TiO2NPs in HEK293 cells; TiO2NPs attenuate transcriptional outputs of TGFβ signaling; TiO2NPs decrease the protein levels of TβRI and TβRII in HEK293 cells; TiO2NPs impair caudal fin regeneration in zebrafish. (PDF)

REFERENCES (1) Antonucci, A.; Kupis-Rozmyslowicz, J.; Boghossian, A. A. Noncovalent Protein and Peptide Functionalization of Single-Walled Carbon Nanotubes for Biodelivery and Optical Sensing Applications. ACS Appl. Mater. Interfaces 2017, 9, 11321-11331. (2) Yao, G.; Li, J.; Chao, J.; Pei, H.; Liu, H.; Zhao, Y.; Shi, J.; Huang, Q.; Wang, L.; Huang, W.; Fan, C. Gold-Nanoparticle-Mediated Jigsaw-Puzzle-Like Assembly of Supersized Plasmonic DNA Origami. Angew. Chem. Int. Ed. Engl. 2015, 54, 2966-2969. (3) Pei, H.; Liang, L.; Yao, G.; Li, J.; Huang, Q.; Fan, C. Reconfigurable Three-Dimensional DNA Nanostructures for the Construction of Intracellular Logic Sensors. Angew. Chem. Int. Ed. Engl. 2012, 51, 9020-9024. (4) Ye, D. K.; Zuo, X. L.; Fan, C. H. DNA Nanostructure-Based Engineering of the Biosensing Interface for Biomolecular Detection. Prog. Chem. 2017, 29, 36-46. (5) Huang, L.; Tao, K. X.; Liu, J.; Qi, C.; Xu, L. M.; Chang, P. P.; Gao, J. B.; Shuai, X. M.; Wang, G. B.; Wang, Z.; Wang, L. Design and Fabrication of Multifunctional Sericin Nanoparticles for Tumor Targeting and Ph-Responsive Subcellular Delivery of Cancer Chemotherapy Drugs. ACS Appl. Mater. Interfaces 2016, 8, 6577-6585. (6) Dvir, T.; Bauer, M.; Schroeder, A.; Tsui, J. H.; Anderson, D. G.; Langer, R.; Liao, R.; Kohane, D. S. Nanoparticles Targeting the Infarcted Heart. Nano Lett. 2011, 11, 4411-4414. (7) Chittasupho, C.; Shannon, L.; Siahaan, T. J.; Vines, C. M.; Berkland, C. Nanoparticles Targeting Dendritic Cell Surface Molecules Effectively Block T Cell Conjugation and Shift Response. Acs Nano 2011, 5, 1693-1702. (8) Boyer, P. D.; Ganesh, S.; Qin, Z.; Holt, B. D.; Buehler, M. J.; Islam, M. F.; Dahl, K. N. Delivering Single-Walled Carbon Nanotubes to the Nucleus Using Engineered Nuclear Protein Domains. ACS Appl.



Mater. Interfaces 2016, 8, 3524-3534. (9) Ghosh, S.; Mohapatra, S.; Thomas, A.; Bhunia, D.; Saha, A.; Das, G.; Jana, B.; Ghosh, S. Apoferritin Nanocage Delivers Combination of Microtubule and Nucleus Targeting Anticancer Drugs. ACS Appl. Mater. Interfaces 2016, 8, 30824-30832. (10) Qian, Y.; Qiao, S.; Dai, Y. F.; Xu, G. Q.; Dai, B. L.; Lu, L. S.; Yu, X.; Luo, Q. M.; Zhang, Z. H. Molecular-Targeted Immunotherapeutic Strategy for Melanoma Via Dual-Targeting Nanoparticles Delivering Small Interfering Rna to Tumor-Associated Macrophages. Acs Nano 2017, 11, 9536-9549. (11) Jiang, S.; Eltoukhy, A. A.; Love, K. T.; Langer, R.; Anderson, D. G. Lipidoid-Coated Iron Oxide Nanoparticles for Efficient DNA and Sirna Delivery. Nano Lett. 2013, 13, 1059-1064. (12) Hu, X.; Li, X.; Yin, M.; Li, P.; Huang, P.; Wang, L.; Jiang, Y.; Wang, H.; Chen, N.; Fan, C.; Song, H. Nanodiamonds Mediate Oral Delivery of Proteins for Stem Cell Activation and Intestinal Remodeling in Drosophila. ACS Appl. Mater. Interfaces 2017, 9, 18575-18583. (13) Wang, T.; Wang, L.; Li, X.; Hu, X.; Han, Y.; Luo, Y.; Wang, Z.; Li, Q.; Aldalbahi, A.; Wang, L.; Song, S.; Fan, C.; Zhao, Y.; Wang, M.; Chen, N. Size-Dependent Regulation of Intracellular Trafficking of Polystyrene Nanoparticle-Based Drug-Delivery Systems. ACS Appl. Mater. Interfaces 2017, 9, 18619-18625. (14) Ge, Z. L.; Pei, H.; Wang, L. H.; Song, S. P.; Fan, C. H. Electrochemical Single Nucleotide Polymorphisms Genotyping on Surface Immobilized Three-Dimensional Branched DNA Nanostructure. Sci. China Chem. 2011, 54, 1273-1276. (15) Chen, P.; Pan, D.; Fan, C.; Chen, J.; Huang, K.; Wang, D.; Zhang, H.; Li, Y.; Feng, G.; Liang, P.; He, L.; Shi, Y. Gold Nanoparticles for High-Throughput Genotyping of Long-Range Haplotypes. Nat. Nanotechnol. 2011, 6, 639-644. (16) Han, L.; Xia, J. M.; Hai, X.; Shu, Y.; Chen, X. W.; Wang, J. H. Protein-Stabilized Gadolinium Oxide-Gold Nanoclusters Hybrid for Multimodal Imaging and Drug Delivery. ACS Appl. Mater. Interfaces 2017, 9, 6941-6949. (17) Cho, H.; Alcantara, D.; Yuan, H.; Sheth, R. A.; Chen, H. H.; Huang, P.; Andersson, S. B.; Sosnovik, D. E.; Mahmood, U.; Josephson, L. Fluorochrome-Functionalized Nanoparticles for Imaging DNA in Biological Systems. ACS Nano 2013, 7, 2032-2041. (18) Han, Y.; Li, X.; Chen, H.; Hu, X.; Luo, Y.; Wang, T.; Wang, Z.; Li, Q.; Fan, C.; Shi, J.; Wang, L.; Zhao, Y.; Wu, C.; Chen, N. Real-Time Imaging of Endocytosis and Intracellular Trafficking of Semiconducting Polymer Dots. ACS Appl. Mater. Interfaces 2017, 9, 21200-21208. (19) Andreou, C.; Neuschmelting, V.; Tschaharganeh, D. F.; Huang, C. H.; Oseledchyk, A.; Iacono, P.; Karabeber, H.; Colen, R. R.; Mannelli, L.; Lowe, S. W.; Kircher, M. F. Imaging of Liver Tumors Using Surface-Enhanced Raman Scattering Nanoparticles. Acs Nano 2016, 10, 5015-5026. (20) Pulakkat, S.; Balaji, S. A.; Rangarajan, A.; Raichur, A. M. Surface Engineered Protein Nanoparticles with Hyaluronic Acid Based Multilayers for Targeted Delivery of Anticancer Agents. ACS Appl. Mater. Interfaces 2016, 8, 23437-23449. (21) Wang, B.; Chen, N.; Wei, Y. L.; Li, J.; Sun, L.; Wu, J. R.; Huang, Q.; Liu, C.; Fan, C. H.; Song, H. Y. Akt Signaling-Associated Metabolic Effects of Dietary Gold Nanoparticles in Drosophila. Sci. Rep. 2012, 2, 563. (22) Chen, N.; Wang, H.; Huang, Q.; Li, J.; Yan, J.; He, D.; Fan, C.; Song, H. Long-Term Effects of Nanoparticles on Nutrition and Metabolism. Small 2014, 10, 3603-3611. (23) Vaijayanthimala, V.; Cheng, P. Y.; Yeh, S. H.; Liu, K. K.; Hsiao, C. H.; Chao, J. I.; Chang, H. C. The


Page 24 of 36

Page 25 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Long-Term Stability and Biocompatibility of Fluorescent Nanodiamond as an in Vivo Contrast Agent. Biomaterials 2012, 33, 7794-7802. (24) Zhang, Y.; Wang, Z. Y.; Li, X. J.; Wang, L.; Yin, M.; Wang, L. H.; Chen, N.; Fan, C. H.; Song, H. Y. Dietary Iron Oxide Nanoparticles Delay Aging and Ameliorate Neurodegeneration in Drosophila. Adv. Mater. 2016, 28, 1387-1393. (25) Yi, H. Y.; Wang, Z. Y.; Li, X. J.; Yin, M.; Wang, L. H.; Aldalbahi, A.; El-Sayed, N. N.; Wang, H.; Chen, N.; Fan, C. H.; Song, H. Y. Silica Nanoparticles Target a Wnt Signal Transducer for Degradation and Impair Embryonic Development in Zebrafish. Theranostics 2016, 6, 1810-1820. (26) Yi, H. Y.; Li, X. J.; Wang, Z. Y.; Yin, M.; Wang, L. H.; Aldalbahi, A.; El-Sayed, N. N.; Wang, H.; Chen, N.; Chen, L. N.; Fan, C. H.; Song, H. Y. Nanodiamonds Interfere with Wnt-Regulated Cell Migration and Adipocyte Differentiation in Cells and Embryonic Development in Vivo. Part. Part. Syst. Char. 2017, 34, 1600208. (27) Papageorgiou, A. C.; Beglitis, N. S.; Pang, C. L.; Teobaldi, G.; Cabailh, G.; Chen, Q.; Fisher, A. J.; Hofer, W. A.; Thornton, G. Electron Traps and Their Effect on the Surface Chemistry of Tio2 (110). Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 2391-2396. (28) Ali, M. A.; Srivastava, S.; Mondal, K.; Chavhan, P. M.; Agrawal, V. V.; John, R.; Sharma, A.; Malhotra, B. D. A Surface Functionalized Nanoporous Titania. Integrated Microfluidic Biochip. Nanoscale 2014, 6, 13958-13969. (29) Ali, M. A.; Mondal, K.; Jiao, Y. Y.; Oren, S.; Xu, Z.; Sharma, A.; Dong, L. Microfluidic Immuno-Biochip for Detection of Breast Cancer Biomarkers Using Hierarchical Composite of Porous Graphene and Titanium Dioxide Nanofibers. ACS Appl. Mater. Interfaces 2016, 8, 20570-20582. (30) Song, Y. Y.; Schmidt-Stein, F.; Bauer, S.; Schmuki, P. Amphiphilic Tio2 Nanotube Arrays: An Actively Controllable Drug Delivery System. J. Am. Chem. Soc. 2009, 131, 4230-4232. (31) Popat, K. C.; Eltgroth, M.; La Tempa, T. J.; Grimes, C. A.; Desai, T. A. Titania Nanotubes: A Novel Platform for Drug-Eluting Coatings for Medical Implants? Small 2007, 3, 1878-1881. (32) Chen, X. B.; Selloni, A. Introduction: Titanium Dioxide (Tio2) Nanomaterials. Chem. Rev. 2014, 114, 9281-9282. (33) Zhao, Y.; Howe, J. L. C.; Yu, Z.; Leong, D. T.; Chu, J. J. H.; Loo, J. S. C.; Ng, K. W. Exposure to Titanium Dioxide Nanoparticles Induces Autophagy in Primary Human Keratinocytes. Small 2013, 9, 387-392. (34) Setyawati, M. I.; Tay, C. Y.; Leong, D. T. Mechanistic Investigation of the Biological Effects of Sio(2), Tio(2), and Zno Nanoparticles on Intestinal Cells. Small 2015, 11, 3458-3468. (35) Thiery, J. P. Epithelial-Mesenchymal Transitions in Tumour Progression. Nat. Rev. Cancer 2002, 2, 442-454. (36) Acloque, H.; Adams, M. S.; Fishwick, K.; Bronner-Fraser, M.; Nieto, M. A. Epithelial-Mesenchymal Transitions: The Importance of Changing Cell State in Development and Disease. J. Clin. Invest. 2009, 119, 1438-1449. (37) Huang, D.; Li, X.; Sun, L.; Huang, P.; Ying, H.; Wang, H.; Wu, J.; Song, H. Regulation of Hippo Signalling by P38 Signalling. J. Mol. Cell Biol. 2016, 8, 328-337. (38) Kasai, H.; Allen, J. T.; Mason, R. M.; Kamimura, T.; Zhang, Z. Tgf-Beta1 Induces Human Alveolar Epithelial to Mesenchymal Cell Transition (Emt). Respir. Res. 2005, 6, 56. (39) Thuault, S.; Valcourt, U.; Petersen, M.; Manfioletti, G.; Heldin, C. H.; Moustakas, A. Transforming



Growth Factor-Beta Employs Hmga2 to Elicit Epithelial-Mesenchymal Transition. J. Cell. Biol. 2006, 174, 175-183. (40) Perez, R. E.; Navarro, A.; Rezaiekhaligh, M. H.; Mabry, S. M.; Ekekezie, II. Trip-1 Regulates Tgf-Beta1-Induced Epithelial-Mesenchymal Transition of Human Lung Epithelial Cell Line A549. Am. J. Physiol. Lung Cell Mol. Physiol. 2011, 300, L799-807. (41) Ye, H.; Wu, Q.; Zhu, Y.; Guo, C.; Zheng, X. Ginsenoside Rh2 Alleviates Dextran Sulfate Sodium-Induced Colitis Via Augmenting Tgfbeta Signaling. Mol. Biol. Rep. 2014, 41, 5485-5490. (42) Jazwinska, A.; Badakov, R.; Keating, M. T. Activin-Beta a Signaling Is Required for Zebrafish Fin Regeneration. Curr. Biol. 2007, 17, 1390-1395. (43) Franzen, P.; Tendijke, P.; Ichijo, H.; Yamashita, H.; Schulz, P.; Heldin, C. H.; Miyazono, K. Cloning of a Tgf-Beta Type-I Receptor That Forms a Heteromeric Complex with the Tgf-Beta Type-Ii Receptor. Cell 1993, 75, 681-692. (44) Wrana, J. L.; Attisano, L.; Carcamo, J.; Zentella, A.; Doody, J.; Laiho, M.; Wang, X. F.; Massague, J. Tgf-Beta Signals through a Heteromeric Protein-Kinase Receptor Complex. Cell 1992, 71, 1003-1014. (45) Attisano, L.; Wrana, J. L. Signal Transduction by the Tgf-Beta Superfamily. Science 2002, 296, 1646-1647. (46) Derynck, R.; Zhang, Y. E. Smad-Dependent and Smad-Independent Pathways in Tgf-Beta Family Signalling. Nature 2003, 425, 577-584. (47) Zavadil, J.; Bottinger, E. P. Tgf-Beta and Epithelial-to-Mesenchymal Transitions. Oncogene 2005, 24, 5764-5774. (48) Thuault, S.; Tan, E. J.; Peinado, H.; Cano, A.; Heldin, C. H.; Moustakas, A. Hmga2 and Smads Co-Regulate Snail1 Expression During Induction of Epithelial-to-Mesenchymal Transition. J. Biol. Chem. 2008, 283, 33437-33446. (49) Medici, D.; Hay, E. D.; Olsen, B. R. Snail and Slug Promote Epithelial-Mesenchymal Transition through Beta-Catenin-T-Cell Factor-4-Dependent Expression of Transforming Growth Factor-Beta3. Mol. Biol. Cell 2008, 19, 4875-4887. (50) Larsen, J. E.; Nathan, V.; Osborne, J. K.; Farrow, R. K.; Deb, D.; Sullivan, J. P.; Dospoy, P. D.; Augustyn, A.; Hight, S. K.; Sato, M.; Girard, L.; Behrens, C.; Wistuba, II; Gazdar, A. F.; Hayward, N. K.; Minna, J. D. Zeb1 Drives Epithelial-to-Mesenchymal Transition in Lung Cancer. J. Clin. Invest. 2016, 126, 3219-3235. (51) Dennler, S.; Itoh, S.; Vivien, D.; ten Dijke, P.; Huet, S.; Gauthier, J. M. Direct Binding of Smad3 and Smad4 to Critical Tgf Beta-Inducible Elements in the Promoter of Human Plasminogen Activator Inhibitor-Type 1 Gene. EMBO J. 1998, 17, 3091-3100. (52) Abdollah, S.; MaciasSilva, M.; Tsukazaki, T.; Hayashi, H.; Attisano, L.; Wrana, J. L. T Beta Ri Phosphorylation of Smad2 on Ser(465) and Ser(467) Is Required for Smad2-Smad4 Complex Formation and Signaling. J. Biol. Chem. 1997, 272, 27678-27685. (53) Liu, X. D.; Sun, Y.; Constantinescu, S. N.; Karam, E.; Weinberg, R. A.; Lodish, H. F. Transforming Growth Factor Beta-Induced Phosphorylation of Smad3 Is Required for Growth Inhibition and Transcriptional Induction in Epithelial Cells. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10669-10674. (54) Schertel, C.; Huang, D.; Bjorklund, M.; Bischof, J.; Yin, D.; Li, R.; Wu, Y.; Zeng, R.; Wu, J.; Taipale, J.; Song, H.; Basler, K. Systematic Screening of a Drosophila Orf Library in Vivo Uncovers Wnt/Wg Pathway Components. Dev. Cell 2013, 25, 207-219.


Page 26 of 36

Page 27 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(55) Chen, Y. G. Endocytic Regulation of Tgf-Beta Signaling. Cell Res. 2009, 19, 58-70. (56) Zhu, Y.; Eaton, J. W.; Li, C. Titanium Dioxide (Tio2) Nanoparticles Preferentially Induce Cell Death in Transformed Cells in a Bak/Bax-Independent Fashion. PLoS One 2012, 7, e50607. (57) Okayasu, I.; Hatakeyama, S.; Yamada, M.; Ohkusa, T.; Inagaki, Y.; Nakaya, R. A Novel Method in the Induction of Reliable Experimental Acute and Chronic Ulcerative-Colitis in Mice. Gastroenterology 1990, 98, 694-702. (58) Williams, K. L.; Fuller, C. R.; Dieleman, L. A.; DaCosta, C. M.; Haldeman, K. M.; Sartor, R. B.; Lund, P. K. Enhanced Survival and Mucosal Repair after Dextran Sodium Sulfate-Induced Colitis in Transgenic Mice That Overexpress Growth Hormone. Gastroenterology 2001, 120, 925-937. (59) Hahm, K. B.; Im, Y. H.; Parks, T. W.; Park, S. H.; Markowitz, S.; Jung, H. Y.; Green, J.; Kim, S. J. Loss of Transforming Growth Factor Beta Signalling in the Intestine Contributes to Tissue Injury in Inflammatory Bowel Disease. Gut 2001, 49, 190-198. (60) Beck, P. L.; Rosenberg, I. M.; Xavier, R. J.; Koh, T.; Wong, J. F.; Podolsky, D. K. Transforming Growth Factor-Beta Mediates Intestinal Healing and Susceptibility to Injury in Vitro and in Vivo through Epithelial Cells. Am. J. Pathol. 2003, 162, 597-608.



Figure 1 TiO2NPs block TGFβ-induced epithelial-mesenchymal transition in A549 cells. (a) A scheme for the process of TGFβ-induced EMT. (b) The effects of TGFβ signal on the cell morphology in the absence or presence of pre-incubated TiO2NPs. Representative cell morphologies were outlined in dotted boxes. Scale bar: 20 µm. (c,d) Protein levels of E-cadherin and N-cadherin analyzed by western blot and quantification. Data represented as mean ±SD (n=4). Student’s t-test, ns means not significant, ***p

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