Therefore we have developed a candidate therapeutic humanized antibody, ANT1034, that may have the potential to be more efficacious and less immunogenic than the current standard anti-CD52 therapy.Anti-CD52 therapy has been shown to be effective in the treatment of a number of B cell malignancies, hematopoietic disorders and autoimmune diseases (including rheumatoid arthritis and multiple sclerosis); however the current standard of treatment, the humanized monoclonal antibody alemtuzumab, is associated with the development of anti-drug antibodies in a high proportion of patients. In order to address this problem, we have identified a novel murine anti-CD52 antibody which has been humanized using a process that avoids the inclusion within the variable domains of non-human germline MHC class II binding peptides and known CD4+ T cell epitopes, thus reducing its potential for immunogenicity in the clinic. The resultant humanized antibody, ANT1034, was shown to have superior binding to CD52 expressing cells than alemtuzumab and was more effective at directing both antibody dependent and complement dependent cell cytotoxicity. Furthermore, when in the presence of a cross-linking antibody, ANT1034 was more effective at directly inducing apoptosis than alemtuzumab. ANT1034 also showed superior activity in a SCID mouse/human CD52 tumour xenograft model where a single 1 mg/Kg dose of ANT1034 led to increased mouse survival compared to a 10 mg/Kg dose of alemtuzumab. Finally, ANT1034 was compared to alemtuzumab in  CD52 is a glycosylphosphatidylinositol (GPI) anchored low molecular weight glycoprotein  found inin vivo cell killing is unclear, in vitro studies have revealed that upon binding to the cell surface CD52, alemtuzumab induces cell destruction via activation of complement dependent cytotoxicity (CDC) ..in vivo in vivo and has demonstrated significant activity against a number of B cell malignancies, particularly in refractory and relapsed chronic lymphocytic leukemia (CLL) for which it was previously marketed under the trade name Campath®/Campath-1H, as well as other non-malignant hematopoietic disorders 14].[14].in v.[14].in isorders 16]17] [17] in v [17] in isorders 19] and  and in visorders 21]. Mos. Mosin visorders 23]..in vivo However, despite its clear successes, alemtuzumab has also been shown to result in substantial toxicity due to attendant immunosuppression associated with its use, and in particular, increased risk of viral and other opportunistic infections 25]26].[26].25][.[26].25]in silico MHC class II binding prediction tools and databases of CD4+ T cell epitopes   per well in AIM-V® for 6 hours before harvesting onto filter mats ) using a TomTec Mach III cell harvester. Counts per minute (cpm) for each well were determined by Meltilex™ scintillation counting on a 1450 Microbeta Wallac Trilux Liquid Scintillation Counter  in paralux, low background counting. Counts per minute for each antibody sample were normalised to the media only control. Results were expressed as a Stimulation Index (SI) defined as the ratio of cpm for the test antibody against a medium-only control using an emperical threshold of SI equal to or greater than 1.9 (SI≥1.90) for positive T cell responses. The threshold of SI ≥ 1.9 has been previously established whereby test samples inducing responses above this threshold are deemed positive. Extensive assay development and previous studies have shown that this is the minimum signal-to-noise threshold allowing maximum sensitivity without detecting large numbers of false positive responses or omitting subtle immunogenic events. For proliferation (n = 6), positive responses were defined by statistical and empirical thresholds as follows:For T cell proliferation assays, 1x10p < 0.05) of the response by comparing cpm of test wells against medium control wells using unpaired two sample Student’s t-test.Significance , where SI = mean of test wells (cpm) / baseline (cpm). Data presented in this way is indicated as SI ≥ 1.90, in vitro maintained lymphoid-derived tumour cells have been shown to express CD52, however the level of expression can vary significantly and the stability of the expression is unpredictable. To overcome this heterogeneity, CD52 expressing cell lines were analysed for levels of CD52 expression and CD52 high expressing (designated ++) clones were identified for Raji, REH and CD52-NS0 cell lines (Many ll lines .Mice were immunised using a combination of CD52 peptide (conjugated to KLH), HUT-78 cells and murine NS0 cells expressing full length human CD52 . The mouse with the highest anti-peptide ELISA titres was sacrificed and derived spleen cells immortalised by fusion with myeloma cells. Hybridomas were selected for supernatant binding activity to CD52 using both a peptide ELISA  and a cell based ELISA . Several hybridomas were identified that bound CD52 either as peptide or on cells however, one hybridoma in particular, clone 2E8, was identified as secreting antibody that strongly bound CD52 in both assay formats. Selected assay positive hybridomas were analysed further by flow cytometry on CD52-NS0 and untransfected NS0 cells .2E8 antibody variable domain genes were amplified, cloned and sequenced  and analThe heavy and light chain V region sequences of the 2E8 murine monoclonal antibody were subcloned into expression vectors containing human IgG1 heavy and kappa light chain constant regions. The resultant chimeric antibody was expressed in, and purified from 293-c18 cells. The binding of the chimeric antibody was compared to alemtuzumab (produced in the same way) in both a direct flow cytometry binding assay and competition assay . The chi++ target cells and it was found that chimeric 2E8 showed significantly enhanced ADCC compared to alemtuzumab  which were synthesised and cloned into human IgG1 / kappa expression vectors. The lead VH and Vκ variant amino acid sequences together with the human antibodies from which sequence segments were sourced are shown in ++ cells by flow cytometry  and for effector function in ADCC and CDC assays , the binding of all of the humanized variants to CD52 peptide was assessed in a competition ELISA against the parent 2E8 chimeric antibody. All lead humanized 2E8 variants displayed competitive binding profiles similar to the parent chimeric antibody ; to discC assays . The chiC assays  and alsoC assays . The hum of 50:1 . Similarmtuzumab . Heat inInterestingly, the cell-based assays were able to discriminate between variants where the peptide competition ELISA could not. In particular, it was observed that the humanized variants containing the VH5 heavy chain, which differs from all other variants by the presence of a tryptophan at position 47 (Kabat) in place of a leucine, showed reduced binding by flow cytometry  and reduin vitro, especially in combination with a cross-linking anti-Fc-antibody, with this effect being mediated by a caspase-dependent pathway. In order to investigate whether the novel CD52 antibody could exert a similar property, the ability of selected humanized anti-human CD52 antibodies to mediate direct cytotoxic effects was assessed using flow cytometry with Annexin V and Propidium Iodide co-staining as markers of apoptosis and necrosis respectively  compared to the isotype matched IgG1 control antibody with ANT1034 eliciting further enhancements in survival, even at the lower dose. Therefore the improved performance of ANT1034 in comparison to alemtuzumab was observed in both in vitro and in vivo models.ANT1034 and alemtuzumab were compared in a Raji human Burkitt lymphoma SCID tumour xenograft mouse model  to measu . Assays for effector function demonstrated that ANT1034 was significantly more effective at killing cells by ADCC and CDC than alemtuzumab and could achieve the same level of cell killing as alemtuzumab with 100x and 10x less antibody in each assay, respectively. One possible mechanism for enhanced cell killing could be due to the increased cell surface decoration (and hence the efficiency of staining cells by flow cytometry) with ANT1034 which may result in improved ADCC responses due to the increased availability of antibody Fc [2 fragment, but in the absence of both effector cells and complement, with greater efficiency than alemtuzumab. During the incubation time used in the assay, ANT1034 induced approximately twice as many cells to become necrotic as alemtuzumab, whilst the numbers of apoptotic cells was broadly similar between the antibodies. ANT1034 was investigated in vivo in a repeat dose CD52+ Burkitt lymphoma mouse xenograft study. ANT1034 produced superior survival rates compared to alemtuzumab even at the lowest dose administered (1 mg/Kg). In order to investigate further the enhanced activity observed for ANT1034 over alemtuzumab, the carbohydrate profile was assessed for each of the two antibodies. Carbohydrate analysis of the two antibodies revealed that they have very similar glycoprofiles Click here for additional data file.S2 Fig(TIF)Click here for additional data file.


The precipitation of excess biliary cholesterol as solid crystals is a prerequisite for cholesterol gallstone formation, which occurs due to disturbed biliary homeostasis. Biliary homeostasis is regulated by an elaborate network of genes in hepatocytes. If unmanaged, the cholesterol crystals will aggregate, fuse and form gallstones. We have previously observed that the levels of osteopontin (OPN) in bile and gallbladder were reduced in gallstone patients. However, the role and mechanism for hepatic OPN in cholesterol gallstone formation is undetermined. In this study, we found that the expression of hepatic OPN was increased in gallstone patients compared with gallstone-free counterparts. Then, we observed that OPN-deficient mice were less vulnerable to cholesterol gallstone formation than wild type mice. Further mechanistic studies revealed that this protective effect was associated with alterations of bile composition and was caused by the increased hepatic CYP7A1 expression and the reduced expression of hepatic SHP, ATP8B1, SR-B1 and SREBP-2. Finally, the correlations between the expression of hepatic OPN and the expression of these hepatic genes were validated in gallstone patients. Taken together, our findings reveal that hepatic OPN contributes to cholesterol gallstone formation by regulating biliary metabolism and might be developed as a therapeutic target for gallstone treatments. Gallstone disease is a major health problem worldwide, and its associated complications and comorbidities impose a substantial financial burden on the health care economy1236910et al. found that OPN-deficient (OPN−/−) mice were completely protected from hepatic insulin resistance which developed in wild type (WT) controls when fed a high-fat diet for 2–4 weekset al. observed that hepatic insulin resistance directly promoted the formation of cholesterol gallstones by increasing the expression of the biliary cholesterol transporters ABCG5 and ABCG8 and decreasing that of the bile acid synthetic enzymes in miceOsteopontin (OPN) is a soluble cytokine and a matrix-associated protein expressed in the majority of tissues and body fluids16In this study, we analysed the correlation between hepatic OPN expression and gallstone formation both in patients and in mice. We reveal that hepatic OPN contributes to cholesterol gallstone formation by regulating biliary metabolism in mice.To investigate the role of hepatic OPN in gallstone formation, we first analysed the expression of OPN in liver tissue samples of GS and GSF by quantitative real-time PCR. The messenger RNA (mRNA) expression of hepatic OPN was higher in GS than in GSF . The resNext, OPN−/− mice were used to further investigate the role and mechanism of hepatic OPN in gallstone formation. When fed a chow diet (CD), neither WT mice nor OPN−/− mice showed crystals or gallstones . Eighty OPN−/− mice fed a CD exhibited increased bile acid levels , organic anion transporter (OATP), ABCB11 or ABCB1a/b  was markedly increased in LD-fed OPN−/− mice. However, the expression of other CYP enzymes was not significantly changed . The expWe next evaluated the expression of 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGCS) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), the two key enzymes in hepatic cholesterol synthesis. The expression of HMGCR was reduced by 40% in OPN−/− mice whereas the expression of HMGCS showed no difference. We then measured two upstream transcriptional factors, sterol regulatory element binding protein 1c (SREBP-1c) and sterol regulatory element binding protein 2 (SREBP-2). The expression of SREBP-2 was reduced in OPN−/− mice whereas the expression of SREBP-1c was not affected . We alsoFinally, we validated the correlations between the expression of hepatic OPN and the related hepatic genes in GS. As expected, the mRNA expression of hepatic OPN had a strong positive correlation with the expression of hepatic SHP, ATP8B1, SR-B1 and SREBP-2 in GS . The resIn this study, we demonstrated that hepatic OPN is involved in biliary homeostasis by regulating the expression of hepatic key genes, and plays an important role in cholesterol gallstone formation.Our previous research revealed that OPN can inhibit cholesterol gallstone formation as an anti-nucleation factor in gallbladder bile, and OPN in gallbladder bile is probably from gallbladder tissues16OPN−/− mice showed an increase in body weight than WT mice when fed with LD for 8 weeks. It may be a result of the reduced expression of hepatic β-KLOTHO in LD-fed OPN−/− mice , which c22232527It has been reported that male C57BL/6J mice are more susceptible to diet-induced gallstones than female C57BL/6J miceIn the gallbladder bile, the cholesterol solubility is maintained by the balance among cholesterol, bile acid and phospholipidAlthough the expression of phospholipid efflux ABCB4 was unchanged, the increased biliary phospholipid content in LD-fed OPN−/− mice might be relevant to the reduced expression of ATP8B1, which plays a role in the transport of phospholipid from the outer to the inner canalicular membrane31Regarding the mechanisms of the increased biliary and hepatic bile acid in LD-fed OPN−/− mice, we measured the hepatic genes involved in the metabolism and transportation of bile acid and found that among the genes measured, only the CYP7A1 gene, the rate limiting enzyme of bile acid synthesisThe expression of CYP7A1 is reciprocally regulated by bile acid via the FXR-SHP pathway and by oxysterols via the LXR pathway. LXR combines with retinoid X receptor (RXR) to form a heterodimer. Then, the LXR–RXR heterodimer promotes the expression of CYP7A1 by binding to the promoter region of CYP7A13639The biliary cholesterol content was reduced whereas the hepatic cholesterol level was increased in LD-fed OPN−/−mice. This phenomenon may be a result of cholesterol transporter dysfunction. Unexpectedly, the expression of ABCG5 and ABCG8 showed no difference between the genotypes. A previous study suggested that pathways independent of ABCG5 and ABCG8 also exist and contribute to cholesterol secretion into bile104648Unexpectedly, we observed an increased content of hepatic TG in LD-fed OPN−/− mice. We measured the expression of the genes involved in hepatic TG metabolism and found that the mRNA and protein levels of apolipoprotein B (ApoB) were decreased in LD-fed OPN−/− mice . ApoB isIn conclusion, we have described a previously unknown function of OPN regulating biliary homeostasis, thus affecting the formation of gallstones both in mice and humans. Our results suggest that new therapeutic strategies designed to modulate OPN activity may be beneficial for preventing the formation of cholesterol gallstones.Human liver tissue samples were collected from 21 GS scheduled for laparoscopic cholecystectomy and 14 GSF undergoing other abdominal operations. All gallstone specimens were classified as cholesterol gallstones because all the stones contained greater than 65% cholesterol. No gallstone was found in any of the GSF controls via ultrasonography. None of the patients had shown any clinical or laboratory evidence of diabetes mellitus, hyperlipoproteinaemia, obesity, alcohol abuse, hepatic carcinoma or other conditions that could affect the function of the liver. In addition, none of them took any medications known to affect lipid metabolism. Informed consent was obtained from all participants prior to enrolment in the study, including permission to collect a liver biopsy. The study protocol was approved by the Ethics Committees of Huashan Hospital, Fudan University. The procedures were performed in accordance with the approved guidelines.C57BL/6J WT mice were purchased from Fudan University . OPN−/− mice in a congenic background were purchased from Jackson Laboratory . WT and OPN−/− mice were fed a CD or LD  for up to 8 weeks. All experiments were performed on mice between 8 and 10 weeks of age (the gender of the mice is shown in The hepatic lipid were extracted into chloroform/methanol (2:1)Total RNA was obtained from tissue with the RNAprep Pure Tissue kit  according to the manufacturer’s instructions. Random primers  were used for the reverse transcription of total RNA to complementary DNA. Quantitative real-time PCR was performed using SYBR Green I chemistry  and the ABI 7900HT Fast Real-Time PCR System . The gene-specific primer sequences are shown in Frozen liver tissue was lysed in ice-cold RIPA. buffer supplemented with protease inhibitors. The protein concentration of the extracts was measured using the BCA Protein Assay Kit . The protein was analysed by 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to poly (vinylidene fluoride) membranes. Anti-CYP7A1 antibodies  were used at a 1:1000 dilution, anti-SR-B1 antibodies  were used at a 1:2000 dilution. A 1:3000 dilution of anti-rabbit immunoglobulin G-HRP  was used as a secondary antibody. After probing individual antibodies, the antigen-antibody complex was visualized using Enhanced Chemiluminescence Supersignal Reagents . The relative average protein level was determined by densitometry. These methods were performed in accordance with the approved guidelines from the Animal Ethics Committee of Fudan University.Formalin-fixed human biopsies were embedded in paraffin and cut into 3 mm sections. Sample slides were incubated with anti-OPN antibodies  at a 1:75 dilution, followed by a 30 min incubation with an HRP-labelled polymer secondary antibody . Sections were viewed with a Nikon ECLIPSE E600 microscope  using 10× objective lenses, and images were acquired with a SPOT INSIGHT™ digital colour camera, model 3.2.0 . Quantification of immunoreactivity was performed on digitally captured colour images saved as TIFF files and analysed using Image-Pro plus 6.0 . Blind immunohistochemistry analysis was conducted by a pathologist of Fudan University. These methods were performed in accordance with the approved guidelines from the Animal Ethics Committee of Fudan University.The data are presented as the mean ± standard deviation (SD) unless otherwise noted. The statistical significance of differences between the means of the experimental groups was evaluated with unpaired Student’s t test, and correlation was performed with Pearson’s test using Prism 6.00 . To meet the criteria of homoscedasticity between variables, the mRNA expression of hepatic OPN was log-transformed, prior to analysis. A difference was considered statistically significant at p < 0.05.How to cite this article: Lin, J. et al. Osteopontin Deficiency Alters Biliary Homeostasis and Protects against Gallstone Formation. Sci. Rep.6, 30215; doi: 10.1038/srep30215 (2016).


Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish.Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia ( This is an especially appealing quality for studies investigating environmental toxicology and physiology where physiological relevance is highly valued.Recent studies demonstrated that human epithelial cells can be effectively immortalized using a combination of Rho-associated kinase (ROCK) inhibitor and mouse 3T3 irradiated feeder layer Oreochromis mossambicus) that are amenable to study the mechanisms of the osmotic stress response. O. mossambicus has emerged as a model fish system for studying the mechanistic basis of the physiological response to salinity challenge, primarily due to its extreme salinity tolerance in culture studies can be informative and physiologically meaningful.As of yet, the application of ROCK inhibitor/feeder layer has not been tested in the immortalization of fish cells. A major goal of this study was to generate immortalized cell lines from Mozambique tilapia (myo-inositol biosynthesis (MIB) pathway, which is a highly inducible component of the response to hyper-saline conditions in several tissues of tilapia, including gill epithelium Myo-inositol is an organic compatible osmolyte that is accumulated in cells when exposed to hyperosmotic stress myo-inositol phosphate synthase (MIPS) and inositol monophosphatase (IMPA), are robustly induced at the mRNA and protein levels following hyperosmotic exposure myo-inositol concentration increases in multiple tissues of tilapia following acclimation to hyperosmotic conditions A number of molecular and physiological mechanisms have been identified as key contributors to the high salinity tolerance observed in tilapia. One example is the in vivo model and is also very specific to osmotic stress.In this study, our goal was to derive several immortalized tilapia cell lines and characterize their osmotolerance and physiological relevance with regard to cellular osmotic stress responses. The MIB pathway was selected for validating the use of cell lines for future studies that investigate response mechanisms to osmotic challenges because it is highly robust in the All animal procedures used in this study were approved by the UC Davis Institutional Animal Care and Use Committee protocol number 13468.O. mossambicus) were kept in de-chlorinated freshwater at 25–27°C in 208 L recirculating tanks at the UC Davis Cole B Animal Facility. The room was kept on a 12 hr∶12 hr (light:dark) photoperiod, and fish were fed daily with a commercial trout pellet diet  at approximately 1% of fish body mass.Adult Mozambique tilapia  medium supplemented with fetal bovine serum (5%), hydrocortisone (0.4 µg/ml), adenine (0.18 mM), insulin (5 µg/ml), transferrin (5 µg/ml), triiodothyronine (20 pM), epidermal growth factor (10 ng/ml)  was used for lip cultures following the initial explant set-up described above using L-15 medium. 10 µM of ROCK inhibitor compound  was also directly added to DMEM/F12 complete medium following the first day. Additionally, lethally irradiated 3T3 cells 2. Cells were passaged every 3–4 days using a 1∶3 splitting ratio.The immortalized TmB cell line Cells of each line  were seeded on plastic coverslips  that were held in 6-well plates  with complete L-15 medium. After two days, coverslips were incubated with Mitotracker Red and Hoechst 33342 live stains  to label mitochondria and nuclei, respectively, and cells were visualized using a fluorescent microscope .Cells of each line  were initially seeded on 12-well plates  and grown to approximately 90% confluency. The day before experiments, cell medium was aspirated and replaced with fresh complete L-15 media. Cells were then exposed to a range of osmolalities in 12-well plates  from baseline medium (315±5 mOsm/kg) to 900 mOsm/kg using 50 mOsm/kg increments by supplementing medium with sodium chloride . One-fifth the total medium volume per well was replaced with a hyperosmotic stock solution to obtain the targeted final osmolality value. Targeted final osmolality was confirmed by taking a small aliquot of medium and measuring the osmolality using an osmometer .Cells were exposed to the different osmolality media and were visually inspected for changes in morphology every 8 hours using an inverted microscope . Percent confluency was also determined at each 8 hour increment within a 24 hour period and was documented with digital images.max) was defined as the concentration at which approximately 50% of the number of live cells remained attached to the substrate of the plastic dish following acute hyperosmotic challenge for 24 hours. The OTmax was extrapolated from the osmotolerance curve for each cell line.A cell viability assay was coupled with visual inspection of osmotolerance (see above) to obtain a quantitative measure for cell survival. At 24 hours, cells grown in a 12-well plates  were washed with 1X PBS, trypsinized, and the cell suspension was diluted (1∶9) in a 0.2 µm filtered electrolytic solution . A blank with Coulter salt solution and medium was used and subtracted to determine the final cell number. Cells plots were obtained for each replicate (n = 3) and cell density in the 10–40 µm size range was quantified. The number of live cells per replicate was quantified using Multisizer3 Coulter Counter software  and values were averaged for each treatment osmolality and used to generate an osmotolerance curve. Maximum acute osmotolerance , OmL (passage 10), and TmB (passage 63) cells were seeded in 6-well plates  and allowed to grow to 80–90% confluency. Hyperosmotic stock solutions were made by supplementing L-15 medium with sodium chloride  and then confirming the osmolality with an osmometer . One-fifth of the total medium in wells (n = 4) was replaced with a hyperosmotic stock solution to reach the target osmolalities of 450 mOsm/kg  and 700 mOsm/kg (maximum acute osmotolerance). Cells were harvested by adding an extraction buffer  supplemented with β-mercaptoethanol to wells of the plate combined with scraping at 4, 8, 16, and 24 hours.OmB cells (passage 11) were seeded in 6-well plates and allowed to grow to ∼70% confluency. Osmolality was increased in increments of 75 mOsm/kg every 24 hours by replacing one-fifth of the medium in wells (n = 4) until the final osmolality  was reached. Cells were then held at the targeted endpoint osmolality for 24 hours after the final osmolality increase and before collection. All cells were harvested using a 10X Tryple solution  at the end of the exposure period and cell pellets were stored at −80°C until analysis.Cells of a tilapia cell line underwent identical volume replacement with iso-osmotic medium (315±5 mOsm/kg) in wells (n = 4) with complete medium to account for handling stress induced in acute and chronic acclimations. Cells were harvested in the wells at 4, 8, 16, and 24 hours for RNA using a lysis extraction buffer .Quantitative real-time PCR (qRT-PCR) was performed to measure mRNA abundance levels of MIPS splice variants  and IMPA isoforms  as described previously t values for MIB enzymes were first normalized against the reference genes (β-actin and 18S RNA) after correcting by efficiency using LinRegPCR software t values were then converted to a fold ratio value using the Pfaffl method, which accounts for differences in primer pair efficiency Target COreochromis niloticus proteome and all available Oreochromis mossambicus sequences, was downloaded from NCBI on March 01, 2014. PEAKS suite 7 was used to double the size of this database by adding a randomly scrambled decoy entry for every sequence and enabled determination of protein false discovery rate (FDR). An FDR of 1% was considered the upper limit for protein identification. Quantitative label-free comparison of OmL and OmB proteomes was performed using PEAKS suite 7 according to manufacturer instructions based on extracted ion chromatogram peak areas of detectable isotopes for unique peptides that identify the corresponding protein . Total ion chromatograms of the corresponding LCMS dataset were used for normalization.OmB (passage 19) and OmL (passage 9) cells were grown in iso-osmotic conditions as described above on 35 mm dishes and harvested when they approached confluency. The proteomes of four technical replicates for each of the two cell lines were analyzed by online LCMS. Proteins were extracted, digested with immobilized trypsin, and buffer exchanged to resuspend tryptic peptides in 3% acetonitrile/97% LCMS-grade water as previously described Analysis of variance (ANOVA) was performed separately on data obtained from each cell line using IMB SPSS software (v. 21). Normality and homoscedasticity of variance were assessed prior to ANOVA using Wilk-Shapiro and Levene tests, respectively. On occasion, a statistically significant outlier was removed to meet the assumption of normality if this could not be achieved through transformation of data. To determine if there was an effect of sampling time on control cells, a one-way ANOVA with time as a factor was performed for all iso-osmotic (315±5 mOsm/kg) samples at every time point or osmolality endpoint. If non-significance was determined (data not shown), all control samples were grouped together in a zero time point. Since variance heteroscedasticity was generally observed in data sets, weighted least squares (WLS) were conducted by grouping samples by the factor of time The OmB cell line was judged to be immortalized after approximately six months of continuous culture. Cells from the original tissue explants divided much faster (1–3 days to confluency) than those in isolated colonies (weeks-months to confluency). Early passaging was generally more successful if tissue pieces from the initial primary culture system were carried over with the trypsinized cells. Alternatively, explants from the initial primary culture could also be regenerated from the original tissue piece by adding medium to the culture dish after trypsinized cells were seeded into new dishes. Small colonies from these trypsinized cells were often visible after early passaging; however, it took several weeks to months before the colonies reached confluency and were ready to be passaged again. Changes in cell morphology were also observed in OmB as cells were continually passaged within the first few months. In the initial stages, cultures appeared to have multiple cell morphologies. The beginning colonies had a combination of star shaped (astrocyte-like) or long and slender (fibroblast-like) cells, followed by an extended period of cell crisis (∼3–4 months). During the cell crisis event, the majority of cells showed signs of deterioration  which led to eventual cell death of these previously viable cells. Cells displaying a typical fibroblast-like phenotype were observed approximately four months post crisis stage and eventually dominated the culture as a continuous cell line after approximately eight successful passages. The initial growth of these cells was poor, but the growth properties of OmB continually improved with further subculturing. These fibroblast-like cells displayed a slender, spindle-like morphology and grew in parallel with neighboring cells to produce characteristic large colonies that resembled waves .The OmL cell line was successfully immortalized using a ROCK inhibitor with 3T3 feeder layer culture after approximately five successful passages. Effective immortalization of lip epithelial-like cells took approximately four months. Passaging was typically unsuccessful for lip epithelial cells without supplementation with ROCK. Epithelial-like cells were observed in the explant outgrowths, and this morphology was maintained. OmL cells were rounded in shape, but elongated cells were occasionally observed in the culture  of each cell line. PCR products were observed for MIPS-160, MIPS-250, IMPA1, IMPA2, β-actin, and 18S rRNA in the expected size ranges.Tilapia-specific primers were tested against cDNA of the three cell lines and PCR products visualized on an agarose gel (data not shown) to verify species origin (1612 proteins and 975 unique protein groups were identified for OmB while 1565 proteins and 1013 unique protein groups were identified for OmL  and S2. max) was not significantly different between OmB (735±2.9 mOsm/kg) and TmB cell lines (706.7±10.9 mOsm/kg). In contrast, the OTmax was significantly higher (p<0.001) in OmL (975±16.1 mOsm/kg) than in the other two cell lines. In the OmB cell line, the number of live cells in treated conditions was significantly lower (p<0.05) than in control conditions (iso-osmotic) beginning at 500 mOsm/kg. The number of live cells was significantly lower in TmB (p<0.001) and OmL (p<0.01) cell lines compared to their respective controls starting at 700 mOsm/kg.Visual inspection following acute osmotolerance testing indicated that cell attachment and confluency decreased with increasing medium osmolality in all tilapia cell lines . Based oUnder 450 mOsm/kg conditions, MIPS-160, MIPS-250, and IMPA1 mRNA expression were significantly upregulated at every time point compared to the control , p<0.001Under 450 mOsm/kg conditions, MIPS-160, MIPS-250, and IMPA1 mRNA expression was significantly upregulated at every time points compared to the control , p<0.001At 450 mOsm/kg, MIPS-160, MIPS-250, and IMPA1 mRNA expression was significantly upregulated at every time points compared to the control , p<0.001in culture correlates to medium osmolality, the OmB cell line was used. A classic dose-dependent response was observed with regard to the mRNA expression of MIPS-160, MIPS-250, IMPA1, and IMPA2 with exposure to higher osmolality , which may explain the eventual domination by fibroblasts In contrast to spontaneously immortalized brain cells (OmB), which transitioned a fibroblast phenotype upon immortalization, the lip cell line (OmL) retained an epithelial-like phenotype after ROCK/feeder layer-induced immortalization. The Rho family of proteins consists of small GTPases that are involved in many intracellular signaling processes including cell polarity, motility, proliferation, and apoptosis In-depth characterization of all cellular properties for each cell line was beyond the scope of this study because our primary goal was to validate the physiological relevance of these cell lines as models for mechanistic studies of osmotic stress responses. Instead, a quantitative proteomic comparison was included in order to identify a few specific cell markers that could differentiate the two newly derived tilapia cell lines . Comparison of OmB and OmL proteomes yielded surprisingly distinct expression of protein biomarkers between the two, which supports the notion that these cell lines are comprised of different cell types. Specifically, OmB expressed higher abundances of proteins that have been documented in brain  protein, neurofilament heavy polypeptide, and optineurin). Astrocytic phosphoprotein PEA-15 is a protein kinase C substrate and is highly abundant in the brain of mammalian systems In contrast, OmL expressed much higher abundances of proteins that are characteristic of epidermis . Keratins are the most abundant structural protein in the cytoplasm of epithelial cells , and as a result, have commonly been used as an epithelial biomarker Phenotypic classification of each cell line based on proteomics data was generally supported by cell morphology observations. Lip epithelium, like fin, has a very high regenerative ability, a property that likely contributed to the cell growth in culture, particularly in the early stages. The OmL cell line displayed an epithelial-like morphology (rounded cell shape), similar to keratinocytes. Interestingly, keratinocytes have been demonstrated to use osmolytes to maintain volume homeostasis in vivo. Cells from euryhaline fish species more frequently encounter osmotic stress in an aquatic environment compared to their terrestrial counterparts and thus may have evolved a higher tolerance to osmotic stress. Nevertheless, mammalian kidney cells, specifically renal inner medulla cells, are also regularly exposed to changes in osmolality due to the urinary concentrating mechanism We found that the osmotolerance observed in the tilapia cells lines is higher than that documented for any other animal cell line. The observed high osmotic tolerance of euryhaline tilapia cells compared to mammalian cells is in agreement with the higher osmotolerance of tilapia myo-inositol contained in the culture media may have an effect on the overall regulation of myo-inositol in the various cell lines via induction of other biochemical pathways. For example, myo-inositol in the media could potentially be fluxed into the cell via various osmolyte co-transporters  and may in turn also have an effect on the regulation of the MIB pathway enzymes. Extracellular myo-inositol and other osmolytes may play an important role in early stages of hyperosmotic acclimation. In the future, it would be interesting to determine if supplementation of media with myo-inositol has any effect on the osmotolerance of the tilapia cell lines. Interestingly, a study by Garcia de Castro and Tunnacliffe It is possible that A recent study used a cichlid astroglial cell line and found that this cell line survived hypo-osmotic challenges and appeared to employ a compensatory regulatory volume decrease myo-inositol. This pathway has been identified as a major component of the response to hyperosmotic challenge in various tissues of tilapia in culture models for studying the mechanisms underlying hyperosmotic MIB pathway induction, we characterized the mRNA expression of the MIB pathway enzymes, MIPS and IMPA. The MIB pathway is a highly conserved pathway in tilapia, across cell types and tissues (in vivo), for responding to hyperosmotic challenge. Specific MIPS splice variants and IMPA isoforms have been found to be highly induced at the mRNA and protein levels by hyperosmotic stress in a number of different tissues in intact tilapia The MIB pathway consists of the enzymes MIPS and IMPA that convert glucose-6 phosphate to the compatible organic osmolyte, in vivo responses to hyperosmotic stress were reproduced in all tilapia cell lines. The same splice variants and isoforms of MIPS and IMPA were induced at the same or even more potency in culture compared to in vivo. It is interesting to note that cells at low  and high passages (TmB: passage 63) are able to utilize the MIPS and IMPA enzymes to respond to osmotic stress. This observation was also made across various putative cell types and reinforces the idea that the MIB pathway is a highly inducible and robust pathway during hyperosmotic stress. At the 450 mOsm/kg  exposure level, there is an earlier (at least 12 hr) maximum of the MIPS mRNA level compared to the 700 mOsm/kg (maximum osmotolerance) exposure level. It is possible that key cellular processes, such as gene expression, are inhibited during severe osmotic stress (700 mOsm/kg), which would explain why there is an observed delay in the transcriptional induction of the MIB pathway enzymes. In the early stages after hyperosmotic stress, cells are recovering from the mechanical stress of shrinkage and intracellular molecular crowding Overall, the previously described in vivoin vivo response of brain tissue in culture is likely a direct result of inhibition of critical cell functions by severe osmotic stress (see above). In contrast, the delay of MIB pathway induction in vivo results from a delay in plasma osmolality increase relative to increased environmental salinity. Brain cells in the whole organism only respond after the signal of increased environmental salinity is reflected in blood plasma osmolality and transduced across the blood-brain barrier to cerebrospinal fluid osmolality. In culture, interfaces such as the body surface or blood-brain barrier are not present and any osmotic change in cell culture medium is more rapidly reflected in responses of cell lines than in cells of intact fish.The MIB enzyme transcriptional response in OmB and TmB was found to be more similar to each other than that of OmL. OmB and TmB showed a greater induction of IMPA relative to MIPS in both 450 mOsm/kg and 700 mOsm/kg conditions. Greater expression of IMPA1 relative to MIPS-160 is similar to what is reported myo-inositol may contribute to osmotic homeostasis in epithelial cells of fish to deal with the more frequent osmolality changes. For example, taurine is known to be an important osmolyte in response to hyperosmotic stress in multiple tissues of tilapia Interestingly, in OmL we found that MIPS splice variant mRNA abundance was higher than that of IMPA isoforms in response to hyperosmotic challenge. It is still unclear why there is greater MIPS mRNA expression in this cell line compared OmB and TmB, but it is thought that conversion of G-6-P to inositol-3-phosphate by MIPS is the rate limiting step of the MIB pathway In summary, we have illustrated that tilapia cell lines generated in this study are useful tools for studying how euryhaline fish respond to osmotic stress. We have clearly demonstrated that tilapia cell lines are highly osmotolerant and possess functional elements of the MIB pathway, which are robustly regulated in response to hyperosmotic challenge in all lines. Thus, this pathway is presumably a highly conserved and important aspect of response to osmotic challenge in tilapia cells. Intricate qualitative differences in the potency of MIB pathway induction and the regulation of MIPS and IMPA in the different cell lines renders the use of multiple tilapia cell lines highly valuable for dissecting mechanistic aspects of MIB pathway regulation during osmotic stress. For example, future work could determine whether knockdown (siRNA) or selective inhibition of MIB enzymes is capable of altering the phenotypic response of the cells . Gene knockdown is rapidly expanding in fish File S1Quantitative protein data for expression of all significantly different proteins in OmB and OmL cells lines. These data correspond to protein data shown on the PEAKS-generated heat map (heat map .(XLSX)Click here for additional data file.File S2Quantitative peptide data for expression of all significantly different proteins in OmB and OmL cells lines. These data are associated with protein data shown on the PEAKS-generated heat map (heat map .(XLSX)Click here for additional data file.File S3Information for all proteins identified in OmB and OmL cell lines. Protein data for each cell line are separated on different tabs.(XLSX)Click here for additional data file.File S4Information for corresponding peptides of all proteins identified in OmB and OmL cell lines. Peptide data for each cell line are separated on different tabs.(XLSX)Click here for additional data file.


Following partial hepatectomy, a coordinated series of molecular events occurs to regulate hepatocyte entry into the cell cycle to recover lost mass. In rats during the first 6 h following resection, hepatocytes are primed by a tightly controlled cytokine response to prepare hepatocytes to begin replication. Although it appears to be a critical element driving regeneration, the cytokine response to resection has not yet been fully characterized. Specifically, the role of one of the key response elements to cytokine signaling (NF-κB) remains incompletely characterized. In this study, we present a novel, genome-wide, pattern-based analysis characterizing NF-κB binding during the priming phase of liver regeneration. We interrogated the dynamic regulation of priming by NF-κB through categorizing NF-κB binding in different temporal profiles: immediate sustained response, early transient response, and delayed response to partial hepatectomy. We then identified functional regulation of NF-κB binding by relating the temporal response profile to differential gene expression. We found that NF-κB bound genes govern negative regulation of cell growth and inflammatory response immediately following hepatectomy. NF-κB also transiently regulates genes responsible for lipid biosynthesis and transport as well as induction of apoptosis following hepatectomy. By the end of the priming phase, NF-κB regulation of genes involved in inflammatory response, negative regulation of cell death, and extracellular structure organization became prominent. These results suggest that NF-κB regulates target genes through binding and unbinding in immediate, transient, and delayed patterns. Such dynamic switch-like patterns of NF-κB binding may govern different functional transitions that drive the onset of regeneration. Liver resection followed by regeneration is used in the clinic for a variety of conditions, including treatment of hepatocellular carcinoma and live liver transplant. Despite its clinical relevance and decades of study, however, the molecular mechanisms governing liver regeneration following resection remain incompletely characterized. In the laboratory, 70% partial hepatectomy (PHx) of rodents has become a standard model to study liver resection and regulation. PHx provides an ideal model to study the liver's regenerative response because the regenerative stimulus is precisely defined and cell proliferation can be studied without the influence of parenchymal injury or excessive inflammatory cell infiltration.Liver regeneration following partial resection typically follows a well-documented set of steps involving hepatocytes and non-parenchymal cells  injection, peaked at approximately 1 h post injection, and gradually decreased activity until beyond 48 h post injection, suggesting that NF-κB acts predominantly during the priming phase of liver regeneration  to identify how NF-κB dynamically binds to genes following PHx in rats. We then related this binding to previously published profiles of gene expression in hepatectomized rats to identify how the dynamic profiles of NF-κB binding relates to regulation of gene expression. Our study demonstrates that NF-κB binding can be classified into dynamic binding switches. Furthermore, these switches appear to dynamically regulate functional pathways associated with liver regeneration. Therefore, NF-κB activation should be interpreted in the context of its dynamic functional binding during liver regeneration.All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at Thomas Jefferson University. Jefferson's IACUC is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care and experiments were designed using the Guide for the Care and Use of Laboratory Animals.Adult (8–10 week old) Sprague-Dawley rats were given ad-libitum access to food (Chow) and water. When their weight reached 275–350 g, they were anesthetized and subjected to 70% PHx by surgical removal of medial and left lateral lobes as per standard procedure  according to manufacturer's instructions. Approximately 50 μg minced liver tissue was fixed for 10 min with 1% formaldehyde, which crosslinks DNA and chromatin binding proteins to ensure co-immunoprecipitation. Glycine (1× in accordance with the EMD Millipore protocol) was then added to quench unreacted formaldehyde. Cells were lysed and chromatin was sheared by sonication to generate fragments of 200–1000 bp . Fragments bound by NF-κB were immunoprecipitated using a ChIP-grade NF-κB antibody  in combination with Protein G conjugated solid support matrix magnetic beads enriched for the antibody of interest using electrophoresis on a 1% agarose gel. This NF-κB antibody has been employed extensively in previous studies to specifically detect NF-κB proteins in Western blots and immunohistochemistry assays, as well as for chromatin immunoprecipitation assays  assays were performed using total liver tissue to map the Purified ChIP DNA was amplified using the GenomePlex Complete Whole Genome Amplification (WGA) kit from Sigma that allows for nearly 500-fold amplification of genomic DNA using OmniPlex Library molecules flanked by universal priming sites. Genome-wide promoter enrichment was measured using the Roche Nimblegen Promoter array platform, which has 720,000 probes . Experimental ChIP and total DNA samples are labeled using 9-mer primers that have Cy3 and Cy5 dyes attached and Klenow added. The labeled experimental ChIP and total DNA samples were co-hybridized to the array for 16–20 h at 420°C, washed, and scanned using an Agilent scanner (Agilent Technologies) following manufacturer instructions.2-ratio data, and peaks were mapped to the nearest gene's transcription start site.Array images were used for data extraction as paired files; genomic feature format files were then produced for analysis of scaled log2-ratio data. The intensity ratio of immunopreciptated to total DNA (not taken through immunoprecipitation steps) was calculated at each genomic position to identify regions where increased signal  was observed relative to the control sample. Peak regions identified as statistically significant binding sites were generated from the scaled logRoche NimbleGen arrays were used because their proprietary, light-mediated synthesis process produces high-density microarrays of long oligonucleotide probes (50–75 mer). These long oligo arrays, when used in combination with high-stringency hybridization protocols, produce results of unparalleled sensitivity and specificity. In addition, because Roche NimbleGen performs ChIP-chip experiments using a two-color protocol, where control and test samples are co-hybridized to the same array, inter-array variation is eliminated. As a result, NimbleGen ChIP-chip service can readily detect enrichment as low as 2-fold of the target binding site in a ChIP sample.www.geo.ncbi.org.Data generated from these experiments as well as processed data used to draw the conclusions of this study were deposited in the Gene Expression Omnibus repository and are publically available at The possible binding regimes (peaks) that correspond to the binding targets were detected if 4 or more probes showed a signal above the cutoff value ranging from 90 to 15% using a 500 bp sliding window. Cut-off values were set as a fraction of the hypothetical maximum signal . An empirical false discovery rate (FDR) was calculated for each peak using a bootstrapping method, which randomized the probe signals 20 times. The calculated FDR is an approximation of the probability of a false positive. To minimize false positives while maximizing NF-κB binding signal, an FDR cutoff of 0.05 was used for all analyses  of the gene is shorter than a threshold cutoff. We defined these “gene regions” as spanning from 5 kb upstream of the TSS to 1.5 kb downstream of the end of transcription. The peak files were annotated with Ensembl version 5.0 (Rnor_5.0) transcript genes using a 5000 base pair cutoff distance from the TSS using the Chip Peak Anno Bioconductor package in R  dynamic binding patterns where NF-κB was identified as bound or unbound at each time: 000, 001, 010, 011, 100, 101, 110, 111. Each pattern was then described by both a binary string  and its digital representation . Because Pattern 0 contains no NF-κB bound genes, it was not considered in the subsequent analyses. Each gene bound by NF-κB was sorted into its binding pattern. Because the patterns are mutually exclusive, each binding pattern of each gene can match only one binding pattern.We used a dynamic pattern-based strategy to analyze the dynamic NF-κB binding post-PHx. We first discretized NF-κB binding by identifying genes as bound (1) or unbound (0) at each time point post-PHx. To be identified as bound, NF-κB binding had to be seen in 2 out of 3 biological replicates at a time point (FDR ≤ 0.05). Additionally, we analyzed how our choice of FDR cutoff would influence our pattern-based analysis and found that choice of FDR cutoff has minimal effect on the fraction of NF-κB bound genes in each of several binding pattern Figure . We founGSE54673)   using default parameters and a string length of 10 , 2440 genes bound at 1 h post-PHx, and 2396 genes bound at 6 h post-PHx (using an FDR cutoff ≤0.05), although, as the priming phase progressed, the numbers of genes bound by NF-κB decreased. Thus, although NF-κB has been shown to become activated quickly following PHx and maintain its activity until at least the end of the priming phase, the overall number of genes bound by NF-κB during the priming phase of liver regeneration remained similar in our analysis. What changed, however, was the distance from the binding site to the transcription start site (TSS) of the nearest gene Figure . Taken tActivation of pro-inflammatory cytokines during the priming phase is a dynamic process involving multiple regulatory feedbacks. Therefore, we used a dynamic pattern-based strategy to analyze the binding of NF-κB to target genes following PHx. We first discretized NF-κB binding by identifying genes as bound 1) or unbound (0) at each time point post-PHx. To be identified as bound, NF-κB binding had to be seen in 2 out of 3 biological replicates at a time point (FDR ≤ 0.05). Because discretized NF-κB binding has 2 distinct states, we were then able to organize NF-κB binding into 7 dynamic binding patterns  Figure . SimilarWe found that immediately post-PHx NF-κB stopped regulating genes associated with multiple pathways and instead began to regulate genes associated with only a few pathways, predominantly with the functions (GO Terms) negative regulation of cell growth and inflammatory response Figure . This rePotentially using this same pool of available NF-κB, NF-κB transiently switched from regulating genes associated with amino acid binding, ion transport, and response to wounding, and began to regulate lipid biosynthetic processes, cell projection, circadian rhythm, and induction of apoptosis Figure . This suAfter there has been sufficient time to begin producing additional NF-κB protein, NF-κB binding undergoes a delayed response switch Figure . As oppoNF-κB also displayed constitutive binding to a select set of genes throughout the priming phase Figure . These gNF-κB binding motif analysis revealed that NF-κB had multiple cofactors that may act to help regulate transcription following PHx Figure . We founWhen analyzed at each time point instead of in each pattern, we found multiple transcription factors that may act in concert with NF-κB including IRF-1, IRF-2, IRF-7, ICSBP (IRF-8), HSF, and MEF-2 Figure . The IRFIn addition, we found several motifs to be selectively enriched at 6 h post-PHx Figure . These mOur motif analysis found several motifs enriched in NF-κB binding sites belonging to the AP-1 and ATF transcription factor families. We therefore investigated the dynamics of AP-1 and ATF transcription factor family activities during the priming phase post-PHx. We found that the AP-1 family of transcription factors was strongly activated by 1 h post-PHx in rats and remained activated over the course of the priming phase Figure . This beWe tested a set of 21 of genes through the use of NF-κB ChIP followed by high-throughput qPCR Figure  Spurgeo. We founOur results indicate that the acute challenge of a partial hepatectomy causes NF-κB binding in the liver to operate as a dynamic switch regulating tissue function. The immediate, transient, and delayed NF-κB signaling profiles appear to serve different purposes in driving regeneration. Early post-PHx, NF-κB binding transitioned from governing many functions to governing mainly those functions necessary to set hepatocytes up for entry into the cell cycle. These functions were maintained throughout the priming phase. Transiently post-PHx, NF-κB binding transitioned away from binding genes involved in maintaining tissue function and toward binding genes involved in apoptosis, circadian rhythm, and hepatic stellate cell activation. The transient switch may therefore be involved in synchronizing healthy cells for entry into the cell cycle while inducing damaged cells to commit apoptosis. The transient switch may also ensure proper timing of hepatic stellate cell activation following PHx. As the priming phase ended, NF-κB began to regulate many genes setting up hepatocytes to enter the cell cycle. This switch may indicate a role for NF-κB contributing to hepatocyte entry into the cell cycle as regeneration progresses past the priming phase. Additionally, several genes involving mitochondrial function that are bound and unbound in this switch  may be important to govern the fraction of metabolic demand delegated to regeneration and that delegated to maintenance of tissue function as hepatocytes enter the cell cycle , dynamics of NF-κB binding in the innate immune system may not be detectable in total tissue extracts. Alternatively, it is possible that some of the “constitutively bound” genes still move to different binding sites closer to the TSS, or are otherwise activated, either through combinatorial binding with other factors, or through chromatin accessibility regulation.These results may have implications beyond the field of liver regeneration for understanding carcinogenesis during inflammatory conditions. Barash et al. proposed that chronic inflammation can increase hepatocyte genomic instability and that these genomically unstable hepatocytes can become tumorigenic following liver resection  gives a much more global view of transcription factor binding which includes the promoter regions and other regions within genes (Ho et al., This genome-wide pattern counts analysis revealed several dynamic NF-κB switches which occur post-PHx. Using this technique, we were able to identify dynamics of NF-κB binding within multiple pathways during liver regeneration. Additionally, we were able to identify a subset of genes that may be critical for healthy tissue function. This analysis strategy has wide application when analyzing transcription factor binding and can be used in other contexts as well as to understand dynamic transcriptional regulation.RV conceived and designed the experiments. BP performed the experiments. DC and LK analyzed the data. DC, JH, and RV wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this review we discuss some of the main molecular targets related with tumor cell proliferation and migration as well as their potential contributions to radiation oncology improvements.Much has been investigated to improve the beneficial effects of radiotherapy especially in that case where radioresistant behavior is observed. Beyond simple identification of resistant phenotype the discovery and development of specific molecular targets have demonstrated therapeutic potential in cancer treatment including radiotherapy. Alterations on transduction signaling pathway related with MAPK cascade are the main axis in cancer cellular proliferation even as cell migration and invasiveness in irradiated tumor cell lines; then, for that reason, more studies are in course focusing on, among others, DNA damage enhancement, apoptosis stimulation, and growth factors receptor blockages, showing promising  Neverthotherapy . Amongstotherapy , 6. In cof tumor , 8. Anotof tumor , specifyof tumor , and oneof tumor –13.The knowledge in tumor biology has been increased in the last years due to cellular signaling pathways discovering, mechanistically identified by molecular tools that allowed investigation of an enormous range of possibilities, even as new mathematical models have also been subsidizing radiotherapy enhancement. The understanding and identification of specific molecular targets with significant therapeutic implications in order to develop new strategies for radiotherapy are crucial to improve patient survival without substantially increasing toxicity.Delivery of advanced radiotherapy techniques has taken new approaches to treatment including stereotactic irradiation and intensity-modulated X-rays beam in order to improve outcomes of cancer treatment and reduce damage to normal surrounding tissues . The claRecent investigations have highlighted differential cellular responses when submitted to intensity-modulated radiation fields , particu in vitro assays, demonstrating that gLQ equation is superior to LQ and MT models in predicting cellular death at high doses of radiotherapy [The effects of combined modality treatments are investigated by using mathematical models to predict cell death as an attempt to fit LQ, MT, and gLQ models to experimental data based onotherapy . A signiotherapy . in vitro survival probability data confirms an optimal timing of mixed irradiation/chemotherapy treatments, leading to a development of an accurate spatial and temporal model of tumor progression and cell cycle dynamics [ in vitro assays and also applying chemotherapy or monoclonal antibodies might be contributed to radiotherapy enhancement.New models for radiobiological cell responses have been proposed and one of those is a simple two-parameter, algorithmic model, which captures the essential biological features of irradiation-induced cell death and associated cell cycle delays. This approach estimates directly the underlying irradiation-induced cell survival and was investigated in mammary carcinoma cell line EMT6/Ro where a comparison of estimated underlying cell survival probability withdynamics . It was dynamics –37. AppaA multiplicity of approaches has been investigated in the efforts to enhance ionizing radiation (IR) effects, particularly, signaling cascade involved in cell proliferation. The mitogen-activated protein kinase (MAPK) pathway transduces signals from the cell membrane to the nucleus in response to a variety of different stimuli and participates in various intracellular signaling pathways that control a wide spectrum of cellular processes, including growth, differentiation, and stress responses, and it is known to have a key role in cancer progression . It was 2/M arrest and enhanced proapoptotic effect after combined IR [1 and G2 phases of cell cycle when treatments are carried out together, primarily regulated by changes in the levels of cyclin D1, p27, and p21, suggesting that alterations of cell cycle by inhibiting the mTOR signaling pathway in combination with radiation have favorable outcomes and it is a promising therapeutic modality for bladder cancer [Since PI3K/AKT/mTOR signaling axis controls cell proliferation and survival this pathway has achieved major importance as a target for cancer therapy . It is abined IR . Anotherr cancer .α) was completely abolished by simultaneous treatment of HNSCC cells with cetuximab [The epidermal growth factor receptor (EGFR) is frequently overexpressed in malignant tumors, and its level is correlated with increased cellular radioresistance . One of etuximab . Despiteetuximab –64.β1 and HIF-1α was noticed [The humanized anti-VEGF monoclonal antibody bevacizumab has single agent activity in previously treated and recurrent cervical, ovarian, and colorectal cancer diseases –68, even noticed , and it  noticed , 74, esp2+ concentrations due to a mitotic blocking induced by buffering of nuclear Ca2+ [2+ regulates cell proliferation is not completely understood, there are reports demonstrating that activation of tyrosine kinase receptors (RTKs) leads to translocation of RTKs to the nucleus to generate localized nuclear Ca2+ signaling which are believed to modulate cell proliferation [2+ buffering decreases EGFR expression, and also the radiosensitization effect of association between nucleoplasmic Ca2+ buffering and X-rays in human squamous cell carcinoma A431, preventing ADAM-17 overexpression, induced by IR. Furthermore, this association promoted less tumor cells proliferation and reduced their survival fractions [2+ as a new target to radiosensitize squamous cell carcinoma.Interestingly, another promising target to radiosensitize tumors resistant to irradiation is nucleoplasmic calcium. The role of nuclear calcium in tumor cell proliferation was previously determined in HepG2 cells showing decreased proliferation rate under low nuclear Caear Ca2+ . Even thferation . We wereractions , suggest foci in human cells have remained controversial in spite of evidences for formation of these repair centers in a dose-response nonlinearity manner [ foci, and, in human head and neck cancer cells, improved cytotoxicity with ABT-888 and IR was found, compared to either agent alone [γ-H2AX/p53BP1-positive foci leading to radiosensitization of these cell lines [γ-H2AX foci correlates with p53 functionality and potentially lethal damage repair in human colorectal carcinoma RKO and prostate cancer DU-145 cells [1 monoclonal antibody that specifically targets EGFR in combination with IR [γ-H2AX foci being able to promote intensification of radiosensitivity of malignant cells expressing EGFR and offer potential improving of therapeutic index of radiotherapy [The concept of DNA repair centers and the meaning of radiation-inducedy manner . While Iy manner . As party manner , 81. Somy manner , 83. They manner . When oly manner . Moreovey manner , 87 evennt alone . Inhibitll lines . In the 45 cells , 91. An  with IR . Becauseotherapy . A signiotherapy . In anototherapy .  Table Apoptosis is a programmed cell death that is currently of intense research interest in cancer biology and it was already established that expression levels of Bcl-2 family proteins in tumors can modulate apoptosis, influencing tumor behavior and treatment , and alsRecent investigations have been suggesting autophagy as a cell death pathway that may mediate cancer cells sensitivity to IR even though it could originate a protective mechanism against the treatment itself by removing proteins and organelles that are damaged or, alternatively, produce an effective cell death process . InhibitIrradiation of primary tumor might promote invasion and favor metastasis by upregulating the expression of genes and activating signaling pathways that are involved in migration and motility. One of the principal pathways involved in alteration of migratory activities is PI3K/Akt signaling pathway that has been implicated in driving metastatic phenotype in thyroid , breast Besides being related to proliferation cascade responses, Akt2, one of the specific isoforms of Akt which is downstream of PI3K, plays important role in promotion of cell migration and invasion. In a xenograft model of colorectal cancer, knockdown of Akt2 in KM20 cell line inhibits liver metastasis; the converse is observed when constitutively active Myr-Akt2 is expressed . It has β) is a tumor promoter and potent inducer of EMT, though it can be a tumor suppressor during the initial stage of tumorigenesis. Ionizing radiation is able to enhance expression of TGFβ in various cell lines. This enhancement occurs along with increase of mesenchymal markers and decrease of epithelial markers, as well as alterations in migratory and invasive capabilities of the cells [β activation in vivo and sensitize even nonmalignant mammary epithelial cells to undergo TGFβ-mediated EMT [ in vitro [β is known to be master regulator of EMT, and IR-induced EMT can be reversed by its inhibition; nonetheless other pathways of EMT induction exist as well. Events associated with EMT induced by IR could be reversed through inhibition of TGFβ signaling with TGFβR inhibitor SB431542 as was already found in A549 cell line [κB [αvβ3 in glioma cells [α5β1 in pancreatic cancer [α3β1 is overexpressed after IR, promoting the migration of meningioma cells via focal adhesion kinase and extracellular signal-regulated kinase [α2β1 is selectively upregulated in irradiated lung cancer cells and is required for aggressive phenotype and invasion in 3D collagen gels and such invasiveness is mediated via PI3K/Akt signaling pathway and then invasion speed in vitro can be reduced significantly by PI3K inhibitor LY294002 [αvβ3 integrin is required during its maturation and activation demonstrating localization of active MMP-2 and αvβ3 integrin at the migration front accelerates cancer cell migration [A growing number of studies demonstrate that IR may enhance the migratory and invasive properties of cancer cells via induction of epithelial-mesenchymal transition (EMT). EMT is an embryonic program important for organogenesis in normal development, but its dysfunction can help the survival and dissemination of cancer cells that is characterized by loss of cell-cell contacts, decreased expression of epithelial markers E-cadherin, beta-catenin, and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin, and vimentin. Several transcription factors have been discovered that can initiate and maintain this process, including Snail, Twist, and Zeb . Furtherhe cells , 115. Hoated EMT . In coloin vitro . Moreoveline [κB . Pharmacline [κB  even as line [κB . Cell moma cells  and α5c cancer  is upregd kinase . MoreoveLY294002 . Additioigration , 125.αvβ3 monoclonal antibody 17E6 and αvβ3/αvβ5 specific antagonist EMD121974 (cilengitide) inhibit in vitro matrigel invasion and lung metastasis formation of tumor cells growing in a preirradiated microenvironment [ in vitro, through combination with IR that allows achieving significant decrease of tumor volumes using intracranial model of human meningioma [Pharmacological inhibition of integrin dependent signaling pathways can be assumed as one of the promising approaches for combined therapy with IR. The function-blocking anti-ironment  and Cileningioma .Nonreceptor tyrosine kinase Src is often activated in various types of cancer via mutations or growth factor signaling pathways including insulin-like growth factor-1 receptor (IGFR-1), EGFR, and platelet-derived growth factor receptor (PDGFR). Then, Src plays an important role in focal adhesion disassembly since its expression results in disruption of focal adhesions and stress fibers leading to the loss of adhesion to the extracellular matrix . This Sr in vitro. Additionally, inhibitors of these signaling pathways with specific inhibitors AG1478, U0126, and SB203580 show decrease of uPA levels in both basal and irradiated-IOMM-Lee cells [ in vitro. When transplanted subcutaneously after subsequent irradiation in xenograft model these cells also have greater lung metastatic potential which is MMP-9 dependent and can be reduced by prototypical MMP-9 inhibitor zoledronic acid [ in vitro is shown to suppress cell migration and invasion abilities in A549 and H1299 cancer cell lines, which is accompanied by decreased expression of MMP-2 and MMP-9 in NSCLC cell lines [Enhancement of invasiveness in response to IR can be caused not only by alterations in cancer cell gene expression profile, which increase cell motility and migratory capacities, but also due to modulation of tumor microenvironment. Irradiated tumor microenvironment may exert potential tumor-promoting effects and tumors growing within a previously irradiated bed tend to be more metastatic . Extraceee cells . Some pree cells , 135. Lenic acid . Proteasll lines . Irradiall lines . in vitro, which means that tumor microenvironment is involved in antimetastatic activities of GDC0941 [Pharmacological inhibition of PI3K/Akt signaling pathway has a great therapeutic potential when combined with IR. In thyroid carcinoma cells PI3K inhibitor GDC0941 significantly inhibits lung metastasis in mice bearing irradiated follicular thyroid carcinoma cells. It is of interest that PI3K is not activated in these cells by IR GDC0941 .Many protocols of irradiation therapy involve irradiation of not only the tumor itself but also nonmalignant cells which surround tumor. As a stress stimulus, irradiation changes significantly gene expression profile of these cells and therefore causes modulation of microenvironment . EnhanceThe increased understanding of the molecular processes underlying cellular sensitivity to IR has led to the identification of novel targets for intervention ; combiniThe trend of radiation therapy improvements has been focused on tyrosine kinase cascade, the principal signaling pathway to development of new target molecules whose members play a pivotal role on cellular proliferation as migration and tumor invasiveness mediators. Predictable responses might be achieved based on combined IR with specific target molecules able to inhibit overexpressed proteins after radiation exposure. In addition, molecular targeted therapy based on signal transduction pathway alterations detected in cancer offers a tailored treatment possibility including improvements on radiation therapy. Thereby, uses of radiotherapy according to predictive markers would potentially reduce costly over treatment, improve the treatment risk-benefit ratio and cancer outcomes , providi in vitro studies are necessary even as systematic reviews focusing on radiobiology and broad molecular targets especially against PI3K/AKT/mTOR pathway that is involved on cellular proliferation as cell migration signaling for the purpose of achieving the best outcome associated with IR to the radiotherapy of the future.Some questions arise concerning how to maximize IR effects combined with these new molecular targets as possible strategies including optimization of dosage and radiation schedule leading to management of toxicities. Further


Furthermore, albino (slc45a2) mutants, which completely lack melanosomes, developed abnormal lens reflections similar to the congenital cataract caused by mutation of the myosin chaperon Unc45b, and an in situ spin trapping assay revealed increased oxidative stress in the lens of albino mutants. Finally transplanting a wildtype lens into an albino mutant background resulted in cataract formation. These data suggest that melanosomes in pigment epithelial cells protect the lens from oxidative stress during embryonic development, likely by buffering trace elements.Altered levels of trace elements are associated with increased oxidative stress that is eventually responsible for pathologic conditions. Oxidative stress has been proposed to be involved in eye diseases, including cataract formation. We visualized the distribution of metals and other trace elements in the eye of zebrafish embryos by micro X-ray fluorescence (μ-XRF) imaging. Many elements showed highest accumulation in the retinal pigment epithelium (RPE) of the zebrafish embryo. Knockdown of the zebrafish  Trace elements are essential for normal development and physiology of the organism. These elements are found at the core of functional domains of metalloproteins in nearly all biological pathways. The presence of metal ion transporters with diverse metal affinities and their specific subcellular localization indicate that each metal needs to be actively transported into proper intracellular compartments. For example, certain transition metals  are required in mitochondria for aerobic respiration, heme synthesis and other functions. Mutation of the mitochondrial iron importer Slc25a37  causes hypochromic anaemia and erythroid maturation arrest in zebrafish2O2) is catalysed for example by metal ions such as iron and copper (the Fenton reaction)2.−) and hydroxyl radicals (.OH) which are generated endogenously in living organisms have been considered as a cause of such deleterious post-translational modifications2O2  and the buffering of protein oxidation by antioxidants (i.e. ascorbic acid and reduced glutathione [GSH])Aberrant localisation or accumulation of trace elements can lead to oxidative stress. The formation of hydroxyl radicals (·OH) from hydrogen peroxide HO2 is catTo visualize sub-cellular element distributions in biological tissues, a number of chemical, or genetically encoded, fluorescent indicators have been developed for some elements such as calciumin situ element distribution in the eyes of zebrafish embryos. We found that mature melanocytes in the pigmented epithelial layer of the eye are major fixation loci for many elements. albino mutant embryos that lack melanosomes showed abnormal lens reflections. The cataractous lenses from albino mutants concurrently showed a signature of ectopic lens protein radicals caused by ROS. These results imply that melanosomes contribute to lens integrity by buffering elements that would otherwise cause deleterious lens protein modifications.We report here the We examined the element distribution in zebrafish embryonic eyes by X-ray fluorescence (μ-XRF) imaging . We firsbrown locus protein, tyrosinase-related protein 1a and b (tyrp1a/b). Tyrp1 expression in melanocytes correlates with the onset of melanin biosynthesisU test; p = 0.0158 for Ba, Ca, K, Mn, Sr and Zn; p = 0.0365 for Br; p = 0.0194 for Se; tyrp1a/b morphants, which are characterized by melanosome maturation arrest at the premelanosomal stage -mediated knockdown of the two zebrafish homologues of the mouse in vivo. This labelling-free non-invasive imaging method visualises contrast created at the structural boundaries of different refractive indicessteif mutants, which carry a loss-of-function allele of the myosin chaperone Unc45bunc45b mutants at 4 dpf, clearly indicating cataract formation .We next examined whether impaired melanosome biogenesis would affect the lens. First we analysed the lenses of at 4 dpf . Embryosat 4 dpf  exhibitealbino . Homozygous albino mutants completely lack melanosomes in the RPE with no obvious defects in the other two types of pigment cell types (xanthophores and iridophores)20tyrp1a/b morphants, we found a significantly increased lens reflection in albino mutants at 4 dpf grown in the dark . Thus, evidence from two impaired melanosome biogenesis phenotypes supports the hypothesis that mature melanosomes protect against cataract formation during embryonic development.To further examine whether melanosomes might protect against cataract formation, we examined a melanosome mutant, albino mutants were examined for the presence of oxidative stress by a spin trapping immunoassay using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) labelling to detect macromolecule radicals generated by oxidative stress23albino mutant embryos were kept in fish water in the dark in the presence of 200 mM DMPO from 24- to 96 hpf and were then processed for immunohistochemistry with an anti-DMPO antibody. Wildtype embryos showed staining levels in the lens comparable to background values determined in a negative staining control without antibodies  produced in the lens are suggested to cause irreversible modifications of lenticular proteins that eventually transform them into insoluble forms6tibodies . In contslc45a2  gene function in the lens. We thus performed lens transplantation experiments  mutant and wildtype hosts. The lens transplantation was performed between stage-matched donor and host embryos at 26–30 hpf in two sets of experiments. One lens of the host was completely removed and replaced with a pd49Tg donor lens. Operated embryos were raised in the dark and the presence of abnormal lens reflection was examined at 4 dpf. The pd49Tg donor lens did not show significant lens abnormality in comparison to those of non-transgenic wild type embryos . Transplanted pd49Tg lenses into wildtype host embryos showed comparable lens quality to non-transplanted pd49Tg siblings . This is consistent with the notion that melanosomes have a role in protecting the lens from cataract.Next, we wished to exclude that the formation of cataract were caused by lack of eriments . The donslc45a2 [albino], tyrp1a/b) lead to cataract formation. The lenses of slc45a  mutants showed signs of increased oxidative stress. Transplantations of wildtype lenses result in cataractous changes in transplanted lenses in slc45a2  mutant but not in wild type hosts. Thus, melanosomes appear to protect the lens from cataract formation. We propose that binding of trace elements in melonosomes plays a buffering role which protects the lens from oxidative damage and cataract formation.We analyzed trace element distribution in the zebrafish eye with sub-micrometer image resolution and found that many elements are highly enriched in the pigment epithelial (PE) layer. Intervening with melanin biosynthesis and mature melanosome formation significantly reduced detected element levels and enrichment in the PE, implying mature melanosomes as primary sequestration loci for various elements. Moreover, melanosomes appear to protect the lens from cataract formation: Elimination of melanosomes by inactivation of two different genes , maintenance of permeability to small molecules appears as a key feature for lens transparency. This may leave the lens quite susceptible to ion composition changes in the vitreous body, including changes in metal ion levels. Accordingly, mutations in the iron storage protein FTL (ferritin light polypeptide) and the iron exporter SLC40A1 (solute carrier family 40 [iron-regulated transporter] member 1) are linked with cataract in the disease phenotype of hyperferritinemia-cataract syndrome (OMIM #600886), most probably as a result of increased radical production catalyzed by iron. Our spin trapping immunoassay results thus imply that zebrafish pigment mutants, in which we observed reduced trace element buffering, may experience elevated intraocular free metal ion concentrations. Free or labile metal ions are a source of ROS implicated in human eye disease36albino homozygous embryos needs to be followed into later life, together with a study on how detoxification pathways (notably those regarding redox homeostasis) develop in the zebrafish lens. The contribution of RPE to lens clarity likely diminishes with time as the eye grows and diffusion distances increase. It is very likely that melanosomes of the later developing iris but also skin melanocytes bind trace elements in the same way as was measured here for the melanosomes of the RPE. In fact, preliminary data suggest that trace elements are also highly enriched in melanocytes residing in the skin close to the eye (unpubl.). It was demonstrated in adult mice that iris melanocytes possess a high ability to synthesize melanin, while the RPE showed only low melanin synthesisalbino individuals may have developed a compensatory mechanism for increased ocular oxidative stress due to the lack of PE. Albino guinea pigs showed increased glutathione peroxidase activities in the retina in comparison to pigmented animalsalbino embryos could be related to their development outside of the maternal body, being more susceptible to oxidative stress than mammalian counterparts in a controlled environment.We could not directly prove the presence of increased metal ions in the lens of melanosome deficient embryos. Free metals may escape fixation. However, the correlative evidence is intriguing: Lack of melanosomes leads to loss of trace element binding in the RPE and concomitant increase of oxidative stress and cataract in the lens. The lens transplantation experiments clearly show that the primary cause of the cataract formation does not reside in the lens tissue itself but is the result of factors from the surrounding mutant tissue acting on the lens, - presumably ROS generating metal ions that cannot be buffered in the mutant melanosome-free environment. To further explore the role of melanosomes in the prevention of cataract formation, the impact of the lens opacity that we observed in in vivo and demonstrate a functional coupling of mature melanosomes in PE cells to homeostasis in adjacent tissues. Mature melanosomes may function as ion scavengers to prevent uncontrolled chemical interactions. A PE-mediated melanosome-dependent protective effect most likely also has implications for eye diseases other than cataract such as age-related macular degeneration (AMD), which is associated with accumulation of aberrant melanoliposomes and RPE cell loss. These observations warrant further characterization of melanosome function and ocular trace element distribution in eye disease.In summary, our data provide novel evidence for an antioxidant, protective effect of pigment epithelial cells All zebrafish husbandry and experimental procedures were performed in accordance with the German animal protection standards and were approved by the Government of Baden-Württemberg, Regierungspräsidium Karlsruhe, Germany (Aktenzeichen 35–9185.64).oC as previously describedDanio rerio) of the AB2O2 wildtype strain, as well as unc45bsb60/sb6017, pd49Tg and albino  were used in this study. For μ-XRF imaging the embryos were fixed at the 72 hpf stage with 4% paraformaldehyde/phosphate buffer saline, dehydrated in a graded series  of ethanol, followed by propylene oxide immersion, and embedded in EPON 812 , as describedoC for 16 hours. Thick 10 μm sections were cut with a microtome with a glass knife and recovered on 500 nm thick silicon-nitride (Si3N4) films.Fish were maintained at 28 tyrosinase-related protein 1a/b, combinations of two antisense morpholinos reported previouslyTo knockdown the expression of 3 solution and measured by inductively coupled plasma mass spectrometry  instant ocean® aquarium sea salt dissolved in Milli-Q water . The samples were diluted by 1:10 and 1:100 with 2% HNODMPO and the anti-DMPO monoclonal antibody were obtained from Enzo Life Sciences . Groups of embryos, each composed of 20 embryos, were kept in 500 μl fish water with or without metal toxicants in the presence of 200 mM DMPO from 24- to 96 hpf. The DMPO concentration was determined to achieve a balance between tissue penetration and non-toxicityLiving embryos were anaesthetized by 0.0168% (w/v) MESAB  and embedded into 0.5% (w/v) low melting agarose with the lateral side of the embryo facing toward the objective. A TCS SP5 confocal system with upright microscopy  was used for visualization of the abnormal light reflection in the lens with a 63× water immersion objective (HCX APO L U-V-I 63.0 × 0.90 WATER UV). An acousto-optical beam splitter (AOBS) was set for reflection mode. Images were acquired in xzy scanning mode with a bit-depth of 8-or 16-bit. Three wavelengths  of light from an argon laser source were used as incident light to detect reflection (453–500 nm) with pinhole opening set to 0.75 airy unit.12 phot/s) was used to raster scan the sample (The samples were imaged at the European Synchrotron Radiation Facility (ESRF) nanoimaging endstation ID22NI (later moved to ID16B). An X-ray beam at 17 keV energy focused to an intense spot  during 26–30 hpf according to the procedure2, 5 mM HEPES, pH7.2) for 30 minutes and released from agarose into zebrafish E3 mediumLens transplantation was performed among stage matched donor t-test, or one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD) test on-demand. For datasets with less than 15 embryos or non-normal distribution, either two-tailed analysis with Mann-Whitney U test or permutation testAll statistical tests were conducted with RHow to cite this article: Takamiya, M. et al. Melanosomes in pigmented epithelia maintain eye lens transparency during zebrafish embryonic development. Sci. Rep.6, 25046; doi: 10.1038/srep25046 (2016).