Proteomic Profiling of Human Hepatic Stellate Cell Line LX2

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Proteomic Profiling of Human Hepatic Stellate Cell Line LX2 Responses to Irradiation and TGF-#1 Baoying Yuan, Yuhan Chen, Zhifeng Wu, Li Zhang, Yuan Zhuang, Xiaomei Zhao, Hao Niu, Jason Chia-Hsien Cheng, and ZhaoChong Zeng J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00814 • Publication Date (Web): 29 Nov 2018 Downloaded from http://pubs.acs.org on November 29, 2018

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Journal of Proteome Research

Proteomic Profiling of Human Hepatic Stellate Cell Line LX2 Responses to Irradiation and TGF-β1 Baoying Yuan1, #, Yuhan Chen1,2 #, Zhifeng Wu1, Li Zhang1, Yuan Zhuang1, Xiaomei Zhao1, Hao Niu1, Jason Chia-Hsien Cheng3, Zhaochong Zeng1, *

1Department

of Radiation Oncology, Zhongshan Hospital, Fudan University,

Shanghai 200032, China 2

Department of Radiation Oncology, Nanfang Hospital, Southern Medical University,

Guangzhou 510515, China 3Division

of Radiation Oncology, Departments of Oncology, National Taiwan

University Hospital, Taipei 100, Taiwan

#Baoying

Yuan and Yuhan Chen contributed equally to this work

*Corresponding Author: ZhaoChong Zeng Department of Radiation Oncology, Zhongshan Hospital, Fudan University, No.180 Feng Lin Road, Shanghai 200032, Email: [email protected] Phone: +86-21-6404-1990 Fax: +86-21-6404-8472 1 ACS Paragon Plus Environment

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Abstract Hepatic stellate cells (HSCs) are the main target of radiation damage and primarily contribute to the development of radiation-induced liver fibrosis. However, the molecular events underlying the radiation-induced activation of HSCs are not fully elucidated. In the present study, human HSC line LX2 was treated with X-ray irradiation and/or TGF-β1 and profibrogenic molecules were evaluated. The iTRAQ LC-MS/MS technology was performed to identify global protein expression profiles in LX2 following exposure to different stimuli. Irradiation or TGF-β1 alone increased expression of α-SMA, collagen 1, CTGF, PAI-1 and fibronectin. Irradiation and TGFβ1 cooperatively induced expression of these profibrotic markers. In total, 102, 137, 155 dysregulated proteins were identified in LX2 cell samples affected by irradiation, TGF-β1 or co-treatment, respectively. Bioinformatic analyses showed that the three differentially expressed protein sets were commonly associated with cell cycle and protein processing in endoplasmic reticulum. The expression of a set of proteins were properly validated: CDC20, PRC1, KIF20A, CCNB1, SHCBP, TACC3 upregulated upon irradiation or irradiation and TGF-β1 co-stimulation, whereas SPARC and THBS1 elevated by TGF-β1 or TGF-β1 plus irradiation treatment. Furthermore, CDC20 inhibition suppresses expression of profibrotic markers in irradiated and TGFβ1-stimulated LX2 cells. Detailed data on potential molecular mechanisms causing the radiation-induced HSC activation presented here would be instrumental in developing radiotherapy strategies that minimize radiation-induced liver fibrosis.

Keywords: iTRAQ, Proteomics, Hepatic stellate cell, Irradiation, Hepatic fibrosis 2 ACS Paragon Plus Environment

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Introduction Technological advancements allow radiation therapy to play an increasingly important role in the management of hepatocellular carcinoma patients at all stages1. Radiationinduced liver disease (RILD) remains a major limiting factor to dose escalation, and investigations to better understand the underlying mechanism of RILD are warranted. Based on the time of clinical manifestation, RILD is described in terms of acute and late injury2. Late radiation liver injury characterized histopathologically by depletion of parenchymal hepatocytes, distortion of the lobular architecture, and fibrosis in pericentral and periportal area, is becoming an increasingly serious clinical problem deserving attention in patients with RILD3. The liver normally harbors hepatic stellate cells (HSCs) in the subendothelial space of Disse, as quiescent cells, storing vitamin A-rich lipid droplets in their cytoplasm4. Following liver damage, they undergo “activation,” toward a phenotype characterized by increased proliferation, contractility, and synthesis of extracellular matrix proteins5. Early hepatic stellate cells activation plays an important role in radiation-induced liver injury and ensuing fibrosis6. Collagen 1 and fibronectin are necessary structural components of extracellular matrix (ECM)7. Excessive accumulation of ECM results from increased synthesis or decreased degradation of ECM or both. Connective tissue growth factor (CTGF) is constantly expressed in activated HSCs and mediates synthesis of ECM components8. Plasminogen activator inhibitor-1 (PAI-1; also known as SERPINE 1) plays a pivotal role in ECM degradation9. 3 ACS Paragon Plus Environment


In our previous study, increased collagen 1 and fibronectin was observed in liver specimens from a rat model of radiation-induced liver fibrosis10. Wang et al. also reported increased collagen 1 mRNA levels along with increased sinusoidal deposition of collagen fibrils in mice receiving liver irradiation11,12. In the study by Cheng et al., CTGF expression was upregulated after liver irradiation in rats13. In vitro studies indicate that irradiating rat mesangial cells led to upregulation of fibronectin and PAI1expression14,15. Zhao et al. reported a radiation-induced increase in collagen 1 and PAI-1 gene expression in rat kidney tubule epithelial cells16,17. Transforming growth factor β1 (TGF-β1) is upregulated in the liver following exposure to ionizing radiation, which may play a role in coordinating the response of liver tissue fibrosis after radiation6. Clinically, TGF-β1 expression is enhanced in patients with hepatitis or cirrhosis and these patients are prone to develop RILD18,19. Interestingly, previous studies have reported that radiation and TGF-β1 co-treatment synergistically induce expression of the profibrotic PAI-1 gene20,21. Although irradiation and TGF-β1 have been implicated in the process of activating HSCs, the contribution of each factor individually and the combination of the irradiation and TGFβ1 toward an activation have not been addressed in detail. The proteome, or proteomics, refers to the entire set of proteins of a cell, tissue, or organism under certain conditions22. Rapid technology developments in proteomics allow us to obtain the quantitative and qualitative mapping of the whole proteome. Isobaric tags for relative and absolute quantitation (iTRAQ) combined with multidimensional liquid chromatography (LC) and tandem MS analysis is a widely 4 ACS Paragon Plus Environment

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accepted technology for quantitative proteomic analysis. Eight-plex iTRAQ reagents allows for the identification and relative quantification of up to eight different samples simultaneously23. Ji et al. have reported the iTRAQ-based comparative proteomic analysis of culture-activated rat HSCs24. The study by Azimifar et al. characterized the proteome of isolated HSCs from mouse25. However, profiles of global protein changes of human HSCs in responses to irradiation and/or TGF-β1 have remained elusive. The aim of this study was to analyze the effects of irradiation, TGF-β1 or both on expression of profibrogenic molecules in human HSC line LX2, including alphasmooth muscle actin (α-SMA), collagen 1, CTGF, PAI-1 and fibronectin. The iTRAQ LC-MS/MS technology was employed to identify comprehensive protein expression profiles in LX2 following exposure to irradiation and/or TGF-β1.

Materials and Methods Cell culture and treatment The human HSC line LX2 of low passage was routinely cultured at 37 °C in a humidified atmosphere containing 5% CO2, in Dulbecco’s modified Eagles’s medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, MA, USA), 100 U/mL penicillin and 100 μg/mL streptomycin. LX2 cells were grown to 80-90% confluence and were serum starved 24 hours prior to treatment with irradiation and/or recombinant human TGF-β1 (240-B, R&D Systems, Minneapolis, MN, USA). For irradiation, cells were treated with a single dose of 8 Gy X-ray irradiation delivered by an ONCORTM linear accelerator (Siemens, Munich, Germany) with a photon beam energy of 6 MV at a dose rate of 3 Gy/min. Directly after irradiation, 5 ACS Paragon Plus Environment


the culture medium with or without 2 ng/mL TGF-β1 was added. In all cases, after administration of different stimuli, the cells were then collected at the indicated time points for following experiments. Sample preparation and protein digestion Forty-eight hours after different stimuli, cells were harvested and resuspended in lysis buffer (8 M Urea, 100 mM Tris-HCl (pH 8.0) 10 mM dithiothreitol (DTT), and proteinase inhibitors), and then ultrasonic crushed to extract total proteins. The cell lysates were centrifuged and the supernatant was collected. Protein concentration was measured by Bradford assay. Protein samples (200 μg) were denatured with dithiothreitol at 37°C for 1 h and alkylated with iodoacetamide in the dark for 1h. The samples were loaded onto filter devices and centrifuged. After washing twice with UA (8 M urea in 100mM Tris-HCl, pH 8.0) and three times with 0.5M Triethylammonium bicarbonate buffer volatile buffer (TEAB), the samples were digested with trypsin (enzyme-to-protein ratio of 1:50) at 37°C overnight. The peptide mixtures were desalted and vacuum dried. iTRAQ labeling and LC-MS/MS analysis iTRAQ analysis was carried out at Beijing Bangfei Bioscience Co., Ltd. (Beijing, China). Protein peptides (100 μg) from each trypsin-digested sample were labeled using the iTRAQ Reagent-8plex Multiplex Kit (ABSCIEX, USA) according to the manufacturer's instructions. Reagents 113 and 117 were used to treat biological duplicates from untreated control cells, reagents 114 and 118 to treat biological duplicates from radiation-stimulated cells, reagents 115 and 119 to treat biological 6 ACS Paragon Plus Environment

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duplicates from TGF-β1-stimulated cells, and reagents 116 and 121 to treat biological duplicates from cells following exposure to radiation and TGF-β1. All samples were analyzed with the Orbitrap Fusion Lumos mass spectrometer coupled with an EasynLC (Thermo Scientific) or an Ultimate 3000 (Thermo Scientific) nano liquid chromatography system. Sequence database search and data analysis The raw mass data were searched against the uniprot- human_170221.fasta database for peptide identification and quantification using Proteome Discoverer 1.4 (Thermo Fisher Scientific) with a false discovery rate (FDR) less than 0.01. The following parameters were set to identify the proteins: peptide mass tolerance = ± 15 ppm, fragment mass tolerance = 20 mmu, maximum missed cleavages = 2, Variable modifications: Oxidation (M) and Acetyl (Protein N-term), Fixed modifications: Carbamidomethyl (C), iTRAQ-8plex (N-term), iTRAQ-8plex (K), iTRAQ-8plex (Y). Fold change of ≥ 1.20 or ≤ 0.83 was set as the meaningful cut-off value to designate upregulated and downregulated proteins respectively. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository26 with the data set identifier PXD011242. Bioinformatics analysis of differentially expressed proteins The differentially expressed proteins were submitted to the Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.8 (https://david.ncifcrf.gov/) based on their accession numbers from the UniProt database. Gene Ontology (GO) analysis was carried out with the threshold of p < 0.05 for the following classifications: 7 ACS Paragon Plus Environment


biological process, molecular function, and cellular component. The visualization of enriched GO terms was performed using the GOPlot R package version 1.0.2. Circle plots and chord plots were generated utilizing GOCircle and GOChord respectively, which display the combination of molecules with their associated GO terms and details of enrichment analysis. In addition, we also used the DAVID for converting UniProt protein accession numbers to Entreze gene IDs. Then the Entreze gene IDs of differentially regulated proteins were selected for pathway enrichment analyses in the Kyoto Encyclopedia of Genes and Genomes (KEGG) using KOBAS 3.0 (http://kobas.cbi.pku.edu.cn). Protein-protein interaction (PPI) network construction Protein-protein interaction (PPI) analysis of the differentially expressed proteins was performed using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) database v10.5 (http://string-db.org). All sources of interaction evidence were included for generation of PPI networks, including text mining, experiments, databases, coexpression, neighborhood, gene fusion, and co-occurrence. The PPI networks were subsequently visualized using Cytoscape v3.5.1 to identify the hub genes by Cytohubb plugin and subnetworks by MCODE plugin. Quantitative Real-Time PCR (qRT-PCR) Total RNA was extracted from LX2 cells of each sample using Trizol reagent (Invitrogen, USA) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized from 1 μg total RNA with the Prime Script RT reagent kit (Takara Bio, Japan). Quantitative PCR amplification was carried out on an ABI 7500 8 ACS Paragon Plus Environment

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real-time detection system using SYBRR Premix Ex Taq. (Takara Bio). The expression levels of target genes were analyzed by the comparative method (2-ΔΔCT) and standardized by the house-keeping gene β-actin. The sequences of primers are listed in Table S1. Western blotting Cells were lysed with RIPA buffer supplemented with phenylmethanesulfonyl fluoride (PMSF) and protease inhibitor cocktail. Protein concentrations were measured by the enhanced BCA protein assay kit (Beyotime Institute of Biotechnology, China) using BSA as standard. Equal amounts of protein samples were subjected to sodium dodecyl sulphate (SDS) -polyacrylamide gel electrophoresis. After electrophoresis, the separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes using a constant current of 300 mA. The PVDF membranes were blocked with 5% skim milk in TBST to prevent nonspecific binding. Then the membranes were incubated with primary antibodies overnight at 4°C. After incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 h at room temperature with shaking, the immunoreactive bands were visualized with the chemiluminescence (ECL) detection system. All antibodies used in this study are listed in Table S2. The intensity of each band was quantified by ImageJ software (US National Institutes of Health, Bethesda, MD, USA). Transfection Three siRNA duplexes specific to human CDC20 mRNA, named siCDC20 #1 (GGAGCUCAUCUCAGGCCAU), siCDC20 #2 (CGGCAGGACUCCGGGCCGA) 9 ACS Paragon Plus Environment


and siCDC20 #3 (GCACAGUUCGCGUUCGAGA) were synthesized by Gene Pharma (Shanghai, China). These siRNA duplexes were transfected into LX2 cells using Lipofectamine 3000 reagent (Life Technologies, USA) following the manufacturer’s instructions. Twenty-four hours after transfection, the cells were treated with 8 Gy Xray irradiation and 2ng/ml TGF-β1 for an additional 48 h. Statistical analysis. The quantitative data are expressed as the mean ± standard error of the mean (S.E.M.). Comparison between two or more groups was subjected to a two-tailed Student’s t-test or one-way analysis of variance (ANOVA) when appropriate. P < 0.05 was considered statistically significant. All statistical analyses were performed with the SPSS 19.0 software (Chicago, IL, USA).

Results Effect of irradiation and/or TGF-β1 on gene expression of α-SMA, collagen I, CTGF, PAI-1 and fibronectin We treated the LX2 cells with 8 Gy of X-ray irradiation and/or 2ng/ml recombinant human TGF-β1 and relative mRNA levels of α-SMA, collagen 1, CTGF, PAI-1, fibronectin were measured. As illustrated in Fig. 1A, when the LX2 cells were exposed to a dose of 8 Gy irradiation, increased expression levels of α-SMA, collagen 1, CTGF, PAI-1, fibronectin mRNA was observed within 24 h, with remained increment at 48 h. Treating LX2 cells with TGF-β1 alone also resulted in an enhanced expression of the mRNA levels of α-SMA, collagen 1, CTGF, PAI-1, fibronectin at 24h and this increase was maintained at 48 h. When irradiation plus TGF-β1 were administered to LX2 cells, 10 ACS Paragon Plus Environment

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α-SMA, collagen 1, CTGF, PAI-1 and fibronectin mRNA levels were further increased as compared with controls or cells treated with irradiation only. This additive effect was observed at 24h and 48h. To determine whether this increase in mRNA levels of theses profibrotic genes was accompanied by increases in protein levels, the cell samples 48h after different stimulation were collected and used for Western blot analysis. As shown in Fig. 1B and C, irradiation alone elicited strong elevation in α-SMA, collagen 1, CTGF, PAI-1 protein levels and weak increase in fibronectin protein expression. Markedly increased protein expression levels of α-SMA, collagen 1, CTGF, PAI-1 and fibronectin were observed 48h after treatment with TGF-β1 only. Further, greater enhancement in protein expression of α-SMA, collagen 1, CTGF, PAI-1 and fibronectin were noted when LX2 cells were stimulated with irradiation and TGF-β1 in combination. To investigate whether modulation of TGF-β1 levels occurred as a result of radiation and TGF-β1 stimulation, the expression of TGF-β1 mRNA and the concentration of TGF-β1 in the supernatants were measured. Irradiation alone and combined with TGF-β1 significantly increased TGF-β1 mRNA expression (Figure S1A). Following irradiation treatment at 48 h, TGF-β1 secretion slightly but significantly increased (Figure S1B). TGF-β1 secretion was further enhanced when irradiation was combined to TGF-β1 stimuli (Figure S1B). These results indicated that irradiation and/or TGF-β1 induced HSCs activation and permitted to choose the same experimental conditions for further experiments. Protein identification The iTRAQ-based high throughput quantitative proteomic approach was used to obtain 11 ACS Paragon Plus Environment


a comprehensive view of the protein ensembles affected by irradiation and/or TGF-β1 treatment on LX2. A full list of these differentially expressed proteins is provided in the Table S3. The expression patterns of dysregulated proteins after different stimuli are represented in a volcano plot (Fig. 2). We identified 102 differentially expressed proteins in LX2 after radiation with a dose of 8 Gy. Of these, 50 were upregulated whilst 52 were downregulated by irradiation (Fig. 2A). A total of 137 proteins were found to be altered after treatment with TGF-β1, 74 of which were upregulated and 63 downregulated (Fig. 2B). In total, 155 proteins were found to be dysregulated in LX2 after irradiation and TGF-β1 co-treatment; the expression of 64 proteins was upregulated and that of 91 proteins was downregulated (Fig. 2C). It can be noticed from Fig. 2D that the three treatments share 29 common proteins. The overlaps among the differentially expressed proteins in three groups are listed in Table S3. Bioinformatics analysis of the altered proteins Differentially expressed proteins were analyzed for GO categories “Biological Process” (BP), ‘‘Cellular Component’’ (CC) and “Molecular Function” (MF) using the DAVID bioinformatics tool. The GO analysis of BP indicated that the altered proteins after irradiation were mainly involved in cell division, cell-cell adhesion and G2/M transition of mitotic cell cycle (Fig. 3). The enriched GO BP for the proteins detected to change after TGF-β1 treatment were extracellular matrix organization, cell adhesion, cell migration, cell cycle arrest, and collagen fibril organization (Fig. 4). Enrichment analysis of BP indicated that combination of irradiation and TGF-β1 mainly acted on cell division, cell-cell adhesion, extracellular matrix organization, collagen fibril 12 ACS Paragon Plus Environment

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organization and response to mechanical stimulus (Fig. 5). Additionally, the chord graph depicts the 7 enriched GO terms associated with the corresponding differentially expressed proteins, as identified by GO analysis. More details of these GO terms are highlighted in circle plots. GO analysis of CC and MF after three treatment groups are presented in the Supplemental results. To annotate the dysregulated proteins further, we applied the web-based platform KOBAS 3.0 to generate the enrichment analysis of KEGG pathway maps. Significantly enriched pathways found associated with the differentially expressed proteins after irradiation included: Protein processing in endoplasmic reticulum, Cell cycle and Protein export (Fig. 6). The identified KEGG pathways modified by TGF-β1 treatment in HSCs included PI3K-Akt signaling pathway, extracellular matrix (ECM)-receptor interaction, Protein processing in endoplasmic reticulum and Focal adhesion (Fig. 7). The differentially-abundant proteins induced by synergistic effect of irradiation and TGF-β1 were mapped to several main pathways, including Cell cycle, Protein processing in endoplasmic reticulum and ECM-receptor interaction (Fig. 8). These annotations provide a great deal of valuable resources for understanding the potential biological functions and pathways of these dysregulated proteins. PPI networks Using STRING database v10.5, we constructed the PPI networks of differentially expressed proteins in an attempt to explicit the molecular mechanisms in HSCs following exposure to ionizing radiation, TGF-β1 or both. After removing the disconnected nodes, the data were imported into Cytoscape to visualize and analyze 13 ACS Paragon Plus Environment


PPI networks. In network studies, the term “hub” refers to a node with a great number of physical connections. The centrality parameter “degree” measures the number of a node’s directly connected neighbours. The hubs (proteins with high degrees) in the network represent essential points in biological networks. As illustrated in figures 9B, 10B and 11B, we screened the top 30 proteins with higher degrees as using the ranking method of degree identified by cytohubba. CCNA2, CCNB1, CDC20, PRC1, AURKA, KIF20A, KIF2C, BUB1B, BIRC5 and CENPE are the top ten hubs identified in the network of irradiation group. FN1, COL1A1, SPARC, POTEE, COL3A1, THBS1, ITGAV, COL5A1, COL5A2 and FGB are determined to be the top scorer hub proteins using the ranking method of degree in the network of TGFβ1 group. GAPDH, CCNA2, CCNB1, AURKA, CDC20, BIRC5, HMMR, BUB1B, HIST1H4I and OIP5 are identified as the top ten hubs in the network of combination group. The Molecular Complex Detection (MCODE) algorithm is a method to perform cluster analysis and identify subnetworks of highly interconnected nodes from the PPI network. We used the Plugin MCODE for construction and analysis of subnetworks from the union network by parameters keeping degree cutoff ≥ 2, node score cutoff ≥ 0.2, k-core ≥2 and maximum depth up to 100. Subnetworks with a number of nodes less than 5 were discarded. The selected subnetworks were subjected to KEGG pathway enrichment analysis again using online website KOBAS 3.0 as described in this study. Two subnetwork modules were observed from the PPI network of irradiation group. As displayed in Fig. 9C, pathway enrichment analysis revealed that subnetwork 1 was mainly associated with cell cycle, while subnetwork 2 was mainly related to Protein 14 ACS Paragon Plus Environment

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processing in endoplasmic reticulum and Protein export. Only one subnetwork with 10 nodes and 20 edges was obtained from PPI network of TGF-β1 treatment group (Fig. 10C). Further functional enrichment analysis showed that this subnetwork was significantly associated with Protein digestion and absorption, ECM-receptor interaction, Focal adhesion and PI3K-Akt signaling pathway. The PPI network of cotreatment group contains two subnetwork modules. Subnetwork 1 was enriched in KEGG pathway related to Cell cycle and subnetwork 2 associated with Protein digestion and absorption (Fig. 11C). Validation of the Proteomic Results in LX2 cell line To validate and investigate whether the proteomic changes occurred at a transcriptional level in LX2 cells following specific stimuli, qRT-PCR analysis was performed at 24h to examine gene expression of eight representative proteins: CDC20, PRC1, KIF20A, CCNB1, SHCBP, TACC3 induced by irradiation and SPARC, THBS1 induced by TGF-β1. As shown in Fig. 12A, the mRNA levels of CDC20, PRC1, KIF20A, CCNB1, SHCBP, TACC3 increased by irradiation or combined irradiation and TGF-β1 stimulation but not by TGF-β1 alone. Similarly, TGF-β1 or TGF-β1 plus irradiation treatment significantly enhanced the expression of SPARC and THBS1 mRNA levels. Western blotting was conducted to further evaluate the expression of 8 selected proteins 48h after different stimuli and yielded similar results to qRT-PCR measurements at the mRNA level (Fig. 12B and C). These results were in agreement with the results from iTRAQ-based study, which implied the credibility and reliability of the proteomic high throughput experiments. 15 ACS Paragon Plus Environment


CDC20 inhibition suppresses expression of profibrotic markers in irradiated and TGF-β1-stimulated LX2 cells Given the robust up-regulation of CDC20 in irradiated and TGF-β1-stimulated LX2 cells, we preliminarily evaluated the potential function of CDC20 on profibrogenic phenotype in LX2 cells. Our data show that transfection of siCDC20 #1, siCDC20 #2 or siCDC20 #3 duplex diminished expression of CDC20 protein (Fig. 13A and B). Moreover, knockdown of CDC20 significantly reduced expression of α-SMA, collagen 1, CTGF, PAI-1 and fibronectin (Fig. 13A and B).

Discussion Classically, the development of radiation-induced late liver damage has been regarded as solely a reduction in the number of surviving clonogens of either hepatocytes or vascular endothelial cell populations27. It is apparent that this target-cell hypothesis is overly simplistic. Recent research in cellular and molecular radiobiology has caused a paradigmatic shift from target cells to orchestrated response between several cell types mediated by early activation of cytokine cascades28. It is well known that hepatic nonparenchymal cells, such as Kupffer cells, liver endothelial cells, and HSCs are radio-responsive and release many cytokines after irradiation29-31. HSC activation is a critical cellular event underlying hepatic fibrosis because these cells are the primary source of increased extracellular matrix32. Upon liver injury, these cells activate into αSMA-expressing contractile myofibroblasts, participating in the formation of the fibrous scar. Activation of HSCs is also observed in irradiated human liver areas and could be responsible for radiation-induced liver injury and ensuing fibrosis6. In good 16 ACS Paragon Plus Environment

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agreement with this observation in human liver tissues, the livers of irradiated rats and mice show enhanced expression of α-SMA, a well known activation marker of HSC33,34. TGF-β1 is a multifunctional cytokine that plays a role in multiple biological processes, such as regulation of cell growth, wound healing and synthesis of extracellular matrix components35. Several studies have reported the involvement of TGF-β1 in the initiation, development, and persistence of radiation-induced fibrosis. Expression of TGF-β1 is significantly increased in rat livers at 9 months after irradiation with 30 Gy and the severity of fibrosis is correlated with the magnitude of this increase36. Geraci and Mariano found that enhanced TGF-β1 gene expression subsequent to irradiation is primarily observed in liver nonparenchymal cells. They postulated that radiation-induced increments in liver nonparenchymal cell populations may be responsible for the development of radiation-induced hepatic fibrosis37. Seong et al. showed increased expression of TGF-β1 mRNA occurs from as early as day 1 after irradiation and gradually increases to 3.6-fold at day 2827. In this study, we investigated the effects of irradiation and/or TGF-β1 on profibrotic gene expression in human HSC line LX2. We found that exposure to irradiation or TGF-β1 alone significantly increased α-SMA levels in LX2 cells. The same treatments also elicited elevations in collagen 1, CTGF, PAI-1 and fibronectin mRNA and protein expression. Combined TGF-β1 and irradiation treatment synergistically induced expression of these profibrotic markers. These results suggest that irradiation and/or TGF-β1 treatment can upregulate gene expression involved in ECM synthesis in LX2 cells evaluated in terms of both mRNA and protein levels. 17 ACS Paragon Plus Environment


Go classifications of these altered proteins identified by iTRAQ as a result of different stimuli identified cell adhesion as the common activated BP. This finding is consistent with a previous study showing that activated HSCs develop cell-cell adherens junctions, which are instrumental in contractile activity38. Irradiation or TGFβ1 alone also affected proteins involved in cell cycle. Likewise, KEGG analysis revealed that 5 genes were enriched in cell cycle pathway among the dysregulated proteins after irradiation and TGF-β1 co-treatment. CDC20 knockdown has been shown to upregulate p21 levels39. Overexpression of p21 inhibited cell cycle progression and HSC proliferation, resulting in HSC senescence and restricting liver fibrosis40,41. Similarly, genes involved in cell cycle regulation were found to be altered in subcutaneous fibroblasts postradiation, indicative of an important role for these genes in radiation-induced fibrosis42. Taken together, all these data demonstrate that cell cycle dysregulation would be linked to the pathogenesis of hepatic fibrosis. GO analyses indicated that TGF-β1 or TGF-β1 plus irradiation treatment affects a list of proteins associated with extracellular matrix organization and collagen fibril organization. Concurrent with this, ECM-receptor interaction was significantly enriched according to KEGG pathway analysis. In addition, focal adhesion and PI3K-Akt signaling pathway were also enriched in the differentially expressed proteins after TGF-β1 stimulation only. The focal adhesion kinase-PI3K/Akt signaling pathway has been linked to the development and progression of hepatic fibrosis and to the regulation of several aspects of HSC activation in vitro, including cell proliferation and extracellular matrix deposition43. These enriched biological process categories and KEGG pathways 18 ACS Paragon Plus Environment

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corresponded to increased ECM synthesis, contractility of activated HSCs and are essential for development of radiation-induced liver fibrosis. Moreover, KEGG pathway enrichment analysis showed that the three differentially expressed protein sets were commonly enriched in protein processing in endoplasmic reticulum. The endoplasmic reticulum (ER) is a membranous network of branching tubules and flattened sacs and serves as a site for protein synthesis and folding, calcium signaling, lipid synthesis44. It has been shown that radiation activates unfolded protein response and results in the accumulation of misfolded proteins, triggering the ER stress response45. TGF-β1 has also been shown to induce ER stress and activation of myofibroblasts46. ER stress response has recently been proposed to play an important role in development of liver fibrosis47,48. An enlarged ER may predispose to myofibroblast activation for excessive deposition and folding of extracellular matrix proteins during fibrosis. Based on the PPI networks, we selected eight representative proteins for verification. Our results revealed that expression of CDC20, PRC1, KIF20A, CCNB1, SHCBP, TACC3 were upregulated upon irradiation or combined irradiation and TGFβ1 stimulation, whereas SPARC and THBS1 elevated by TGF-β1 or TGF-β1 plus irradiation treatment. Moreover, CDC20 knockdown suppresses profibrogenic effects of irradiation and TGF-β1 in LX2 cells, implicating a potential role of CDC20 on fibrosis. KIF20A, also known as MKLP2/RAB6KIFL, plays a crucial role in cell proliferation, adhesion and migration49-51. Intriguingly, the fibrotic fibroblasts showed more than two-fold elevations in PRC1, CCNB1 and KIF20A mRNA levels compared 19 ACS Paragon Plus Environment


with normal fibroblasts according to genome-wide expression profiling analysis52. Chen et al. reported that SHCBP1 was a regulator of proliferation induced by fibroblast growth factor signaling in neural progenitor cells53. Transforming acidic coiled-coil protein 3 (TACC3) is a member of the TACC family, essential for spindle assembly, chromosomal function and mitotic progression54. TACC3 is believed to correlate with cell proliferation, transforming capability, migratory behavior and promotes epithelialmesenchymal transition through the PI3K/Akt and ERK signaling pathways55. Ha et al. reported that elevated levels of TACC3 result in the accumulation of DNA doublestrand breaks and alter the normal cellular response to DNA damage56. Cells expressing high levels of TACC3 display increased sensitivity to ionizing radiation56. Secreted protein, acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that is highly expressed in activated HSCs and fibrotic livers57,58. Accumulating evidence highlights that TGF-β1 increased SPARC expression in several types of cells59-61. In agreement with this, we herein show that TGF-β1 treatment of LX2 led to an upregulation of SPARC mRNA and protein expression. On the other hand, previous studies demonstrated that SPARC has a capacity to stimulate TGF-β1 expression in mesangial cells and cultured HSCs62,63. There may be a positive feedback between SPARC and TGF-β1 that intensifies SPARC expression and TGF-β1 activity synergistically. Atorrasagasti et al. found that SPARC knockdown reduced collagen I mRNA expression in both LX2 and rat primary HSCs64. Besides, SPARC was found to induce expression of PAI-1, suggesting its role in inhibiting degradation of ECM deposition58. Moreover, adenovirus-mediated inhibition of SPARC alleviates liver 20 ACS Paragon Plus Environment

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fibrosis in an experimental rat model63. Thrombospondin 1(THBS1; also known as TSP-1) is an adhesive protein that constitutes the ECM and affects diverse cellular activities65. Promotion of THBS1 expression by TGF-β1 was observed in LX2 in the present study. This finding is consistent with a previous study showing that increased secretion of THBS1 from TGF-β1 treated LX2 cells66. THBS1 expression was also increased in stellate cells from carbon tetrachloride induced liver fibrosis67. In addition, Breitkopf et al. showed that THBS1 activates latent TGF-β1 secreted by hepatic stellate cells68. THBS1 and TGF-β1 may engage one another in a reciprocal relationship, contributing to progression of liver fibrosis. Taken together, the findings reported here provide a comprehensive proteome profile of human HSC line LX2 following exposure to radiation and/or TGF-β1 and give clues for further investigation of the mechanisms behind radiation-induced liver fibrosis.

Supporting Information Supplemental Methods Supplemental results about bioinformatics analysis Figure S1: Effect of irradiation and/or TGF-β1 on TGF-β1 mRNA expression and secretion Figure S2: GO terms in molecular function of differentially expressed proteins in irradiation group Figure S3: GO terms in cellular component of differentially expressed proteins in 21 ACS Paragon Plus Environment


irradiation group Figure S4: GO terms in molecular function of differentially expressed proteins in TGFβ1 treatment group Figure S5: GO terms in cellular component of differentially expressed proteins in TGFβ1 treatment group Figure S6: GO terms in molecular function of differentially expressed proteins in irradiation and TGF-β1 co-treatment group Figure S7: GO terms in cellular component of differentially expressed proteins in irradiation and TGF-β1 co-treatment group Table S1: Primer sequences for real-time PCR Table S2: Antibodies used in the present study Table S3: Identification list of proteins in LX2 following exposure to irradiation and/or TGF-β1

Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. U1505229).

Author Contributions Zhaochong Zeng conceived and designed the experiments. Baoying Yuan and Yuhan Chen carried out the plan and wrote this paper. Zhifeng Wu, Li Zhang, Yuan Zhuang, Xiaomei Zhao, Hao Niu and Jason Chia-Hsien Cheng have given many advices and 22 ACS Paragon Plus Environment

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carried out the data analysis. All authors read and approved the final manuscript.

Conflict of interest The authors declare that there is no conflict of interests regarding the publication of this paper.

References 1. Jihye, C.; Jinsil, S. Application of Radiotherapeutic Strategies in the BCLCDefined Stages of Hepatocellular Carcinoma. Liver Cancer, 2012, 1, 216-225. 2. Maity, A.; Kao, G. D.; Muschel, R. J.; McKenna, W. G. Potential molecular targets for manipulating the radiation response. International Journal of Radiation Oncology• Biology• Physics 1997, 37, 639-653. 3. Cromheecke, M.; Konings, A.; Szabo, B.; Hoekstra, H. Liver tissue tolerance for irradiation: experimental and clinical investigations. Hepato-gastroenterology 2000, 47, 1732-1740. 4. Friedman, S. L. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiological reviews 2008, 88, 125-172. 5. Tsuchida, T.; Friedman, S. L. Mechanisms of hepatic stellate cell activation. Nature Reviews Gastroenterology and Hepatology 2017, 14, 397. 6. Sempoux, C.; Horsmans, Y.; Geubel, A.; Fraikin, J.; Van Beers, B.; Gigot, J.; Lerut, J.; Rahier, J. Severe radiation ‐ induced liver disease following localized radiation therapy for biliopancreatic carcinoma: Activation of hepatic stellate cells as an early event. Hepatology 1997, 26, 128-134. 7. Friedman, S. L. Mechanisms of hepatic fibrogenesis. Gastroenterology 2008, 134, 1655-1669. 8. Rachfal, A. W.; Brigstock, D. R. Connective tissue growth factor (CTGF/CCN2) in hepatic fibrosis. Hepatol Res 2003, 26, 1-9. 9. Zhao, W.; Spitz, D. R.; Oberley, L. W.; Robbins, M. E. Redox modulation of the pro-fibrogenic mediator plasminogen activator inhibitor-1 following ionizing radiation. Cancer Res 2001, 61, 5537-5543. 10. Du, S.-S.; Qiang, M.; Zeng, Z.-C.; Zhou, J.; Tan, Y.-S.; Zhang, Z.-Y.; Zeng, H.Y.; Zhong-Shan, L. Radiation-induced liver fibrosis is mitigated by gene therapy inhibiting transforming growth factor-β signaling in the rat. International Journal of Radiation Oncology• Biology• Physics 2010, 78, 1513-1523. 11. Wang, S.; Lee, K.; Hyun, J.; Lee, Y.; Kim, Y.; Jung, Y. Hedgehog signaling influences gender-specific response of liver to radiation in mice. Hepatology international 2013, 7, 1065-1074. 23 ACS Paragon Plus Environment


12. Wang, S.; Lee, Y.; Kim, J.; Hyun, J.; Lee, K.; Kim, Y.; Jung, Y. Potential role of Hedgehog pathway in liver response to radiation. PLoS One 2013, 8, e74141. 13. Cheng, W.; Xiao, L.; Ainiwaer, A.; Wang, Y.; Wu, G.; Mao, R.; Yang, Y.; Bao, Y. Molecular responses of radiation-induced liver damage in rats. Molecular medicine reports 2015, 11, 2592-2600. 14. Wang, J.; Robbins, M. E. Radiation-induced alteration of rat mesangial cell transforming growth factor-β and expression of the genes associated with the extracellular matrix. Radiation research 1996, 146, 561-568. 15. Zhao, W.; O'Malley, Y.; Robbins, M. E. Irradiation of rat mesangial cells alters the expression of gene products associated with the development of renal fibrosis. Radiation research 1999, 152, 160-169. 16. Zhao, Y. O. M., S. Wei, MEC Robbins, W. Irradiation of rat tubule epithelial cells alters the expression of gene products associated with the synthesis and degradation of extracellular matrix. International journal of radiation biology 2000, 76, 391-402. 17. Zhao, W.; Spitz, D. R.; Oberley, L. W.; Robbins, M. E. Redox modulation of the pro-fibrogenic mediator plasminogen activator inhibitor-1 following ionizing radiation. Cancer research 2001, 61, 5537-5543. 18. Annoni, G.; Weiner, F. R.; Zern, M. A. Increased transforming growth factor-β1 gene expression in human liver disease. Journal of hepatology 1992, 14, 259-264. 19. Cheng, J. C.-H.; Wu, J.-K.; Lee, P. C.-T.; Liu, H.-S.; Jian, J. J.-M.; Lin, Y.-M.; Sung, J.-L.; Jan, G.-J. Biologic susceptibility of hepatocellular carcinoma patients treated with radiotherapy to radiation-induced liver disease. International Journal of Radiation Oncology• Biology• Physics 2004, 60, 1502-1509. 20. Hageman, J.; Eggen, B. J.; Rozema, T.; Damman, K.; Kampinga, H. H.; Coppes, R. P. Radiation and transforming growth factor-β cooperate in transcriptional activation of the profibrotic plasminogen activator inhibitor-1 gene. Clinical cancer research 2005, 11, 5956-5964. 21. Niemantsverdriet, M.; de Jong, E.; Langendijk, J. A.; Kampinga, H. H.; Coppes, R. P. Synergistic induction of profibrotic PAI-1 by TGF-β and radiation depends on p53. Radiotherapy and Oncology 2010, 97, 33-35. 22. Graves, P. R.; Haystead, T. A. Molecular biologist's guide to proteomics. Microbiology and molecular biology reviews 2002, 66, 39-63. 23. Boersema, P. J.; Raijmakers, R.; Lemeer, S.; Mohammed, S.; Heck, A. J. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nature protocols 2009, 4, 484. 24. Ji, J.; Yu, F.; Ji, Q.; Li, Z.; Wang, K.; Zhang, J.; Lu, J.; Chen, L.; Qun, E.; Zeng, Y. Comparative proteomic analysis of rat hepatic stellate cell activation: a comprehensive view and suppressed immune response. Hepatology 2012, 56, 332-349. 25. Azimifar, S. B.; Nagaraj, N.; Cox, J.; Mann, M. Cell-type-resolved quantitative proteomics of murine liver. Cell metabolism 2014, 20, 1076-1087. 26. Vizcaino, J. A.; Cote Rg Fau - Csordas, A.; Csordas A Fau - Dianes, J. A.; Dianes Ja Fau - Fabregat, A.; Fabregat A Fau - Foster, J. M.; Foster Jm Fau - Griss, J.; Griss J Fau - Alpi, E.; Alpi E Fau - Birim, M.; Birim M Fau - Contell, J.; Contell J Fau - O'Kelly, G.; O'Kelly G Fau - Schoenegger, A.; Schoenegger A Fau - Ovelleiro, D.; Ovelleiro D 24 ACS Paragon Plus Environment

Page 24 of 41

Page 25 of 41 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


Fau - Perez-Riverol, Y.; Perez-Riverol Y Fau - Reisinger, F.; Reisinger F Fau - Rios, D.; Rios D Fau - Wang, R.; Wang R Fau - Hermjakob, H.; Hermjakob, H. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013. Nucleic acids research, 2012, 41, D1063-D1069. 27. Seong, J.; Kim, S. H.; Chung, E. J.; Lee, W. J.; Suh, C. O. Early alteration in TGF-β mRNA expression in irradiated rat liver. International Journal of Radiation Oncology• Biology• Physics 2000, 46, 639-643. 28. Bentzen, S. M. Preventing or reducing late side effects of radiation therapy: radiobiology meets molecular pathology. Nature Reviews Cancer 2006, 6, 702. 29. Christiansen, H.; Saile, B.; Neubauer-Saile, K.; Tippelt, S.; Rave-Fränk, M.; Hermann, R. M.; Dudas, J.; Hess, C. F.; Schmidberger, H.; Ramadori, G. Irradiation leads to susceptibility of hepatocytes to TNF-α mediated apoptosis. Radiotherapy and oncology 2004, 72, 291-296. 30. Du, S.-S.; Qiang, M.; Zeng, Z.-C.; Ke, A.-W.; Ji, Y.; Zhang, Z.-Y.; Zeng, H.-Y.; Liu, Z. Inactivation of kupffer cells by gadolinium chloride protects murine liver from radiation-induced apoptosis. International Journal of Radiation Oncology• Biology• Physics 2010, 76, 1225-1234. 31. Zhou, L.-Y.; Wang, Z.-M.; Gao, Y.-B.; Wang, L.-Y.; Zeng, Z.-C. Stimulation of hepatoma cell invasiveness and metastatic potential by proteins secreted from irradiated nonparenchymal cells. International Journal of Radiation Oncology• Biology• Physics 2012, 84, 822-828. 32. Puche, J. E.; Saiman, Y.; Friedman, S. L. Hepatic stellate cells and liver fibrosis. Comprehensive Physiology, 2011, 3, 1473-1492. 33. Rave-Fränk, M.; Malik, I. A.; Christiansen, H.; Naz, N.; Sultan, S.; Amanzada, A.; Blaschke, M.; Cameron, S.; Ahmad, S.; Hess, C. F. Rat model of fractionated (2 Gy/day) 60 Gy irradiation of the liver: long-term effects. Radiation and environmental biophysics 2013, 52, 321-338. 34. Wang, S.; Hyun, J.; Youn, B.; Jung, Y. Hedgehog signaling regulates the repair response in mouse liver damaged by irradiation. Radiation research 2012, 179, 69-75. 35. Pohlers, D.; Brenmoehl, J.; Löffler, I.; Müller, C. K.; Leipner, C.; Schultze-Mosgau, S.; Stallmach, A.; Kinne, R. W.; Wolf, G. TGF-β and fibrosis in different organs— molecular pathway imprints. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 2009, 1792, 746-756. 36. Anscher, M. S.; Crocker, I. R.; Jirtle, R. L. Transforming growth factor-β1 expression in irradiated liver. Radiation research 1990, 122, 77-85. 37. Geraci, J.; Mariano, M. Radiation hepatology of the rat: parenchymal and nonparenchymal cell injury. Radiation research 1993, 136, 205-213. 38. Guyot, C.; Lepreux, S.; Combe, C.; Doudnikoff, E.; Bioulac-Sage, P.; Balabaud, C.; Desmoulière, A. Hepatic fibrosis and cirrhosis: the (myo) fibroblastic cell subpopulations involved. The international journal of biochemistry & cell biology 2006, 38, 135-151. 39. Amador, V.; Ge, S.; Santamaría, P. G.; Guardavaccaro, D.; Pagano, M. APC/C Cdc20 controls the ubiquitin-mediated degradation of p21 in prometaphase. Molecular cell 2007, 27, 462-473. 25 ACS Paragon Plus Environment


40. Kong, X.; Feng, D.; Wang, H.; Hong, F.; Bertola, A.; Wang, F. S.; Gao, B. Interleukin ‐22 induces hepatic stellate cell senescence and restricts liver fibrosis in mice. Hepatology 2012, 56, 1150-1159. 41. Zheng, J.; Dong, P.; Mao, Y.; Chen, S.; Wu, X.; Li, G.; Lu, Z.; Yu, F. lincRNA‐ p21 inhibits hepatic stellate cell activation and liver fibrogenesis via p21. The FEBS journal 2015, 282, 4810-4821. 42. Rødningen, O. K.; Børresen-Dale, A.-L.; Alsner, J.; Hastie, T.; Overgaard, J. Radiation-induced gene expression in human subcutaneous fibroblasts is predictive of radiation-induced fibrosis. Radiotherapy and oncology 2008, 86, 314-320. 43. Lopez-Sanchez, I.; Dunkel, Y.; Roh, Y.-S.; Mittal, Y.; De Minicis, S.; Muranyi, A.; Singh, S.; Shanmugam, K.; Aroonsakool, N.; Murray, F. GIV/Girdin is a central hub for profibrogenic signalling networks during liver fibrosis. Nature communications 2014, 5, 4451. 44. Zhang, K.; Kaufman, R. J. From endoplasmic-reticulum stress to the inflammatory response. Nature 2008, 454, 455. 45. Moretti, L.; Cha, Y. I.; Niermann, K. J.; Lu, B. Switch between apoptosis and autophagy: radiation-induced endoplasmic reticulum stress? Cell cycle 2007, 6, 793798. 46. Heindryckx, F.; Binet, F.; Ponticos, M.; Rombouts, K.; Lau, J.; Kreuger, J.; Gerwins, P. Endoplasmic reticulum stress enhances fibrosis through IRE1 α ‐ mediated degradation of miR‐150 and XBP‐1 splicing. EMBO molecular medicine 2016, 8, 729-744. 47. Tanjore, H.; Lawson, W. E.; Blackwell, T. S. Endoplasmic reticulum stress as a pro-fibrotic stimulus. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 2013, 1832, 940-947. 48. Li, X.; Wang, Y.; Wang, H.; Huang, C.; Huang, Y.; Li, J. Endoplasmic reticulum stress is the crossroads of autophagy, inflammation, and apoptosis signaling pathways and participates in liver fibrosis. Inflammation Research 2015, 64, 1-7. 49. Gasnereau, I.; Boissan, M.; Margall-Ducos, G.; Couchy, G.; Wendum, D.; Bourgain-Guglielmetti, F.; Desdouets, C.; Lacombe, M.-L.; Zucman-Rossi, J.; Sobczak-Thépot, J. KIF20A mRNA and its product MKlp2 are increased during hepatocyte proliferation and hepatocarcinogenesis. The American journal of pathology 2012, 180, 131-140. 50. Taniuchi, K.; Furihata, M.; Saibara, T. KIF20A-mediated RNA granule transport system promotes the invasiveness of pancreatic cancer cells. Neoplasia 2014, 16, 10821093. 51. Stangel, D.; Erkan, M.; Buchholz, M.; Gress, T.; Michalski, C.; Raulefs, S.; Friess, H.; Kleeff, J. Kif20a inhibition reduces migration and invasion of pancreatic cancer cells. journal of surgical research 2015, 197, 91-100. 52. Shi-Wen, X.; Renzoni, E. A.; Kennedy, L.; Howat, S.; Chen, Y.; Pearson, J. D.; Bou-Gharios, G.; Dashwood, M. R.; du Bois, R. M.; Black, C. M. Endogenous endothelin-1 signaling contributes to type I collagen and CCN2 overexpression in fibrotic fibroblasts. Matrix Biology 2007, 26, 625-632. 53. Chen, J.; Lai, F.; Niswander, L. The ubiquitin ligase mLin41 temporally promotes 26 ACS Paragon Plus Environment

Page 26 of 41

Page 27 of 41 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


neural progenitor cell maintenance through FGF signaling. Genes & development 2012, 26, 803-815. 54. Ohoka, N.; Nagai, K.; Hattori, T.; Okuhira, K.; Shibata, N.; Cho, N.; Naito, M. Cancer cell death induced by novel small molecules degrading the TACC3 protein via the ubiquitin–proteasome pathway. Cell death & disease 2014, 5, e1513. 55. Ha, G.-H.; Park, J.-S.; Breuer, E.-K. Y. TACC3 promotes epithelial–mesenchymal transition (EMT) through the activation of PI3K/Akt and ERK signaling pathways. Cancer letters 2013, 332, 63-73. 56. Ha, G.; Kim, J.; Petersson, A.; Oh, S.; Denning, M.; Patel, T.; Breuer, E. TACC3 deregulates the DNA damage response and confers sensitivity to radiation and PARP inhibition. Oncogene 2015, 34, 1667. 57. Frizell, E.; Liu, S. L.; Abraham, A.; Ozaki, I.; Eghbali, M.; Sage, E. H.; Zern, M. A. Expression of SPARC in normal and fibrotic livers. Hepatology 1995, 21, 847-854. 58. Nakatani, K.; Seki, S.; Kawada, N.; Kitada, T.; Yamada, T.; Sakaguchi, H.; Kadoya, H.; Ikeda, K.; Kaneda, K. Expression of SPARC by activated hepatic stellate cells and its correlation with the stages of fibrogenesis in human chronic hepatitis. Virchows Archiv 2002, 441, 466-474. 59. WRANA, J. L.; OVERALL, C. M.; SODEK, J. Regulation of the expression of a secreted acidic protein rich in cysteine (SPARC) in human fibroblasts by transforming growth factor β. The FEBS Journal 1991, 197, 519-528. 60. Reed, M. J.; Vernon, R. B.; Abrass, I. B.; Sage, E. H. TGF ‐ β 1 induces the expression of type I collagen and SPARC, and enhances contraction of collagen gels, by fibroblasts from young and aged donors. Journal of cellular physiology 1994, 158, 169-179. 61. Abe, K.; Hibino, T.; Mishima, H.; Shimomura, Y. The cytokine regulation of SPARC production by rabbit corneal epithelial cells and fibroblasts in vitro. Cornea 2004, 23, 172-179. 62. Francki, A.; Bradshaw, A. D.; Bassuk, J. A.; Howe, C. C.; Couser, W. G.; Sage, E. H. SPARC regulates the expression of collagen type I and transforming growth factorβ1 in mesangial cells. Journal of Biological Chemistry 1999, 274, 32145-32152. 63. Camino, A. M.; Atorrasagasti, C.; Maccio, D.; Prada, F.; Salvatierra, E.; Rizzo, M.; Alaniz, L.; Aquino, J. B.; Podhajcer, O.; Silva, M. Adenovirus‐mediated inhibition of SPARC attenuates liver fibrosis in rats. The journal of gene medicine 2008, 10, 9931004. 64. Atorrasagasti, C.; Aquino, J. B.; Hofman, L.; Alaniz, L.; Malvicini, M.; Garcia, M.; Benedetti, L.; Friedman, S. L.; Podhajcer, O.; Mazzolini, G. SPARC downregulation attenuates the profibrogenic response of hepatic stellate cells induced by TGF-β1 and PDGF. American Journal of Physiology-Gastrointestinal and Liver Physiology 2011, 300, G739-G748. 65. Pal, S. K.; Nguyen, C. T. K.; Morita, K. i.; Miki, Y.; Kayamori, K.; Yamaguchi, A.; Sakamoto, K. THBS1 is induced by TGFB1 in the cancer stroma and promotes invasion of oral squamous cell carcinoma. Journal of oral pathology & medicine 2016, 45, 730-739. 66. Shimada, H.; Staten, N. R.; Rajagopalan, L. E. TGF-β1 mediated activation of Rho 27 ACS Paragon Plus Environment


kinase induces TGF-β2 and endothelin-1 expression in human hepatic stellate cells. Journal of hepatology 2011, 54, 521-528. 67. Smalling, R. L.; Delker, D. A.; Zhang, Y.; Nieto, N.; Mcguiness, M. S.; Liu, S.; Friedman, S. L.; Hagedorn, C. H.; Wang, L. Genome-wide transcriptome analysis identifies novel gene signatures implicated in human chronic liver disease. American Journal of Physiology-Gastrointestinal and Liver Physiology 2013, 305, G364-G374. 68. Breitkopf, K.; Sawitza, I.; Westhoff, J.; Wickert, L.; Dooley, S.; Gressner, A. Thrombospondin 1 acts as a strong promoter of transforming growth factor β effects via two distinct mechanisms in hepatic stellate cells. Gut 2005, 54, 673-681.

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Fig. 1 Radiation and/or TGF-β1 upregulated expression of α-SMA, collagen I, CTGF, PAI-1 and fibronectin. (A) qRT-PCR analysis of α-SMA, collagen I, CTGF, PAI-1, and fibronectin mRNA levels. (B, C) Representative western blots showed the expression of α-SMA, collagen I, CTGF, PAI-1 and fibronectin in LX2 cells. RT: irradiation treatment. * P < 0.05 vs. control group; # P < 0.05 vs. RT group.

Fig. 2 Volcano plot illustrates differentially abundant proteins in irradiation group (A), TGF-β1 treatment group (B), and co-treatment group (C). The -log10 (P) is plotted against the log10 (FC). The non-axial vertical lines denotes 0.83 or 1.2 fold change. FC: fold change. (D) Venn chart presents the overlaps among the differentially expressed proteins in three groups.



Fig. 3 GO terms in biological process of differentially expressed proteins in irradiation group (A). The x axis shows the counts of proteins involved in each term. Chord graph (B) representing the relationship between differentially expressed proteins and their related GO terms. The color code represents the log2 fold change, with red corresponding to upregulation and blue corresponding to downregulation. Circle plot (C) highlighting protein expression differences within each GO term, with red dots depicting upregulated proteins and blue dots showing downregulated proteins. The height of the inner rectangle represents the P value of the GO term and the color corresponds to Z score (representing the overall change of each individual term): blue, decreased; red, increased.


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Fig. 4 GO terms in biological process of differentially expressed proteins in TGF-β1 treatment group; same legend as in Figure 3.



Fig. 5 GO terms in biological process of differentially expressed proteins in irradiation and TGF-β1 co-treatment group; same legend as in Figure 3.


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Fig. 6 KEGG pathway analysis for the differentially expressed proteins after irradiation. (A), Enriched pathways based on web-based platform KOBAS 3.0. (B), Visualization of the proteins to pathway interactions using the Cytoscape software. Blue nodes represent pathways, red nodes represent upregulated proteins and green nodes represent downregulated proteins.



Fig. 7 KEGG pathway analysis for the differentially expressed proteins after TGF-β1 treatment. (A), Enriched pathways based on web-based platform KOBAS 3.0. (B), Visualization of the proteins to pathway interactions using the Cytoscape software; same legend as in Figure 6.


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Fig. 8 KEGG pathway analysis for the differentially expressed proteins after irradiation and TGF-β1 co-treatment. (A), Enriched pathways based on web-based platform KOBAS 3.0. (B), Visualization of the proteins to pathway interactions using the Cytoscape software; same legend as in Figure 6.



Fig. 9 PPI network of differentially expressed proteins in irradiation group (A). Red nodes represent upregulated proteins, green nodes represent downregulated proteins. The size and color of the nodes are proportional to the expression fold change. (B) Top 30 proteins with higher degrees identified by cytohubba. (C) Subnetworks obtained by MCODE and associated KEGG pathways.


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Fig. 10 PPI network of differentially expressed proteins in TGF-β1 treatment group; same legend as in Figure 9.



Fig. 11 PPI network of differentially expressed proteins in irradiation and TGF-β1 cotreatment group; same legend as in Figure 9.


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Fig. 12 Validation of proteomic results by qRT-PCR and western blotting. (A) qRTPCR analysis of CDC20, PRC1, KIF20A, CCNB1, SHCBP, TACC3, SPARC and THBS1 mRNA levels. (B, C) Representative western blots showed the expression of CDC20, PRC1, KIF20A, CCNB1, SHCBP, TACC3, SPARC and THBS1 in LX2 cells. RT: irradiation treatment. * P < 0.05 vs. control group.

Fig. 13 LX2 cells were transfected with control or three different CDC20 siRNA 39 ACS Paragon Plus Environment


duplexes (siCDC20 #1, siCDC20 #2 or siCDC20 #3) for 24h, irradiated with 8 Gy Xray and incubated with 2ng/ml TGF-β1 for an additional 48 h. (A, B) Representative western blots showed the expression of CDC20, α-SMA, collagen I, CTGF, PAI-1 and fibronectin. * P < 0.05 vs. control group.


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Proteomic Profiling of Human Hepatic Stellate Cell Line LX2

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