Abstract
The obesity epidemic is developing into the most costly health problem facing the world. Obesity, characterized by excessive adipogenesis and enlarged adipocytes, promotes morbidities such as diabetes, cardiovascular disease and cancer. Regulation of adipogenesis is critical to our understanding of how fat cell formation causes obesity and associated health problems. Thy1 (also called CD90), a widely used stem cell marker, blocks adipogenesis and reduces lipid accumulation. Thy1 knockout-mice are prone to diet-induced obesity. While the importance of Thy1 in adipogenesis and obesity is now evident, how its expression is regulated is not. We hypothesized that DNA methylation plays a role in promoting adipogenesis and affects Thy1 expression. Using the methylation inhibitor 5-aza-2’-deoxycytidine (5-aza-dC), we investigated whether DNA methylation alters Thy1 expression during adipogenesis in both mouse 3T3-L1 pre-adipocytes and mouse mesenchymal stem cells. Thy1 protein and mRNA levels were decreased dramatically during adipogenesis. However, 5-aza-dC treatment prevented this phenomenon. Pyrosequencing analysis shows that the CpG sites at the Thy1 locus are methylated during adipogenesis. These new findings highlight the potential role of Thy1 and DNA methylation in adipogenesis and obesity.
Introduction
Obesity rates have risen markedly in the last 30 years. More than 700 million people worldwide are clinically obese1,2. Obesity promotes type 2 diabetes, fatty liver disease, and cardiovascular disease, and is linked with certain cancers3-5. Health care costs associated with obesity and its comorbidities are enormous and will continue to rise as currents trends continue and the population ages6. Thus, a further understanding of obesity and its underlying mechanisms and causes are urgently needed.
Obesity results from a positive energy balance when more calories are consumed than are used. The surplus energy is packaged into lipid-based storage molecules and sent to fat storage cells called adipocytes. In obesity, there is both an increase in adipocyte size and an increase in adipocyte number to accommodate the lipid7. Adipocytes are formed during the process of adipogenesis and arise from stem cells, fibroblasts, or other progenitor cells when appropriately programmed8. Adipogenesis is a highly regulated process that requires the activation of several key signaling pathways, including STAT5 and Fyn and activation of the transcription factors PPARγ and C/EBPα. Numerous genes involved in fatty acid transport and storage, such as fatty acid binding protein 4 (Fabp4) are induced during adipogenesis to promote lipid accumulation in adipocytes.
Several proteins including Pref-1, Wnt, TGFβ, and Thy1 (formally called CD90) have been shown to inhibit adipogenesis by blocking pro-adipogenic signaling9-13. Our recent study showed that Thy1 blocked the activity of the Src family kinase, Fyn, in pre-adipocytes14. Thy1 mediated inhibition of Fyn activity prevented adipocyte formation. Interestingly, while pre-adipocytes expressed high levels of Thy1, its expression was lost during adipogenesis, and mature adipocytes expressed almost no Thy1. Thy1 is a member of the immunoglobulin supergene family and is a glycophosphatidyl inositol linked surface protein. While Thy1 is expressed on pre-adipocytes and subsets of fibroblasts, neurons, and stem cells, little is known about how its expression is controlled. We recently showed that Thy1 levels can be regulated by microRNAs. Specifically, the miR-103/107 family of miRNAs can target Thy1 mRNA and reduce its expression15. Furthermore, the Thy1 gene contains several CpG rich elements termed CpG islands, which are hotspots for cytosine methylation and gene regulation16,17. To date, there have been no reports studying DNA methylation of Thy1 during adipogenesis. However, Thy1 methylation has been studied in context of fibrosis and in T cells, where they show an increase in Thy1 methylation correlated with a decrease in Thy1 expression18-20. Therefore, we investigated the same CG rich region within intron 1 (termed Thy1-CGI1), which is part of the promoter17,20-23.
While changes in DNA methylation patterns at the Thy1 locus have not been characterized in the context of adipogenesis, recent reports have shown that DNA methylation changes are an integral part of adipocyte formation24,25. In the most widely used and well accepted model of adipogenesis, the murine 3T3-L1 pre-adipocyte line, global DNA methylation has been shown to increase during adipogenesis26,27. Interestingly, addition of the DNA methylation inhibitor 5-aza-2’-deoxycytidine (5-aza-dC) at the onset of adipogenic differentiation can completely block adipocyte formation, suggesting that DNA methylation changes are essential for adipogenesis27,28. Specific changes in regional DNA methylation are also critical for adipogenesis, and blocking these patterns can alter the differentiation pathway of precursor cells towards osteoblastogenesis rather than adipogenesis24,29,30. For example, when blocking DNA methylation using 5-aza-dC in 3T3-L1 cells, Wnt10a expression increases through the hypomethylation of several of its CpG sites. The increase in Wnt10a expression helps steer the differentiation program towards osteoblastogenesis and away from adipogenesis30.
Because Thy1 protein and mRNA levels are rapidly suppressed during adipogenic differentiation, we hypothesized that its reduced expression is linked to hypermethylation of the Thy1 locus. Excitingly, we are the first to show methylation sensitive pyrosequencing data of the Thy1-CGI1 region and report herein that DNA methylation is one of the essential regulators of Thy1 expression during adipogenesis.
Results
Reduced Thy1 expression during adipogenesis is partially attenuated by 5-aza-dC in bone marrow-derived mouse mesenchymal stem cells
Previously, we demonstrated that pre-adipocytes and mesenchymal stem cells must down-regulate Thy1 expression to differentiate into adipocytes14. To determine if DNA methylation regulates Thy1 expression during adipogenesis, we first pharmacologically inhibited DNA methyltransferase in bone-marrow derived mouse mesenchymal stem cells (mMSCs) before differentiation into adipocytes28. mMSCs were treated daily with either DMSO (vehicle) or the DNA methyltransferase inhibitor, 5-aza-dC for 12 days and were concurrently treated with either media alone or with the adipogenic cocktail medium (ACT), starting at day 3 (Fig. 1A). mMSCs typically differentiate into adipocytes in 2 weeks when exposed to ACT31. Therefore, we examined an intermediate time point to examine Thy1 levels. As expected, when given ACT, Thy1 levels decreased while fatty acid binding protein 4 (Fabp4) levels were increased compared to media alone (Fig. 1B-D); Fabp4 is highly expressed by adipocytes and serves as an adipogenic marker. However, inhibiting DNA methylation with 5-aza-dC increased Thy1 protein and mRNA levels versus media alone and attenuated the down-regulation of Thy1 in ACT samples (Fig. 1B-C). We also determined that protein and mRNA levels of Fabp4, a protein highly expressed by adipocytes that serves as an adipogenic marker, were decreased in mMSCs treated with 5-aza-dC (Fig. 1B,D). Immunofluorescence staining revealed that compared to cells not treated with ACT, 5-aza-dC-treated mMSCs had increased surface expression of Thy1 and decreased Fabp4 levels (Fig. 1E). These results indicate that inhibition of DNA methylation impairs adipogenesis.
Reduced Thy1 expression, which is necessary for adipogenesis, is prevented by inhibition of global DNA methylation in 3T3-L1 cells
Primary mesenchymal stem cells are heterogeneous with only some cells fully differentiating into adipocytes32. Thus, we next used the well-established pre-adipocyte murine cell line, 3T3-L1. Previously, we showed that fully differentiated adipocytes treated with ACT no longer expressed Thy1 protein and mRNA33. To observe changes in Thy1 expression, we examined an intermediate point in adipogenesis. At only four days of treatment with ACT the cells are only partially differentiated, and Fabp4 expression increases. Cells were treated daily for 7 days with either DMSO or 5-aza-dC and starting at day 3 were either given ACT or continued with media alone (Fig. 2A). As expected, Thy1 decreased at both the protein and mRNA level in cells given ACT compared to media alone (Fig 2B-C). However, we show that treatment with 5-aza-dC prevents the decrease in Thy1 mRNA levels in ACT-treated cells, while media alone with 5-aza-dC causes no significant changes (Fig 2C). 5-aza-dC also caused a decrease in Fabp4 levels in ACT-treated cells, indicative of decreased adipogenesis (Fig 2B,D). Inhibiting global methylation blunts adipogenesis, which is reflected by the decreased levels of Fabp4 expression and in part sustains Thy1 levels.
5-aza-dC partially restores Thy1 cell surface expression in 3T3-L1 cells when exposed to adipogenic cocktail
We next examined Thy1 cell surface expression on 3T3-L1 cells, since Thy1 is a known cell marker on pre-adipocytes and is readily detected via flow cytometry and immunofluorescence. Cells were treated for 7 days daily with either DMSO or 5-aza-dC, while ACT samples were given the adipogenic cocktail starting at day 3, as previously described. The representative histogram in Figure 3A shows cells treated with ACT shift out of the Thy1+ gate into Thy1-during differentiation, while cells treated with ACT and 5-aza-dC mostly remain in the Thy1+ gate. As expected, pre-adipocytes treated with ACT expressed significantly less surface Thy1 than cells with media alone, as evidenced by a lower mean fluorescence intensity (MFI) (Fig 3B). However, pre-adipocytes cultured with ACT and 5-aza-dC showed a significant increase in Thy1 MFI compared to ACT alone, which occurred in tandem with an increase in the percentage of Thy1-positive cells (Fig 3B-C). Using immunofluorescence, we confirmed that Thy1 expression was sustained in ACT-treated pre-adipocytes also treated with 5-aza-dC, while Fabp4 expression decreased compared to cells treated with ACT alone (Fig. 3D).
Thy1(CD90) gene expression is regulated by DNA methylation during adipogenesis
We went on to examine Thy1 DNA methylation, which typically occurs in cytosine (CG) rich regions and is commonly associated with gene silencing34,35. As the transcriptional activation of the Thy1 gene involves both the promoter and intron 1 in some cell types21,23, and previous publications have referred to intron 1 as part of the promoter17,19-23, we focused on a CpG rich region within intron 1 that we termed Thy1-CGI1 (Fig 4A). Using a pyrosequencer, we analyzed methylation levels of 5 consecutive CpG sites within Thy1-CGI1. 3T3-L1 pre-adipocytes were treated as described previously. We found that Thy1-CGI1 is hypermethylated during differentiation comparing the average DNA methylation of ACT-treated samples to those treated with media alone. Treatment with 5-aza-dC resulted in reduced methylation of these CpG sites in the Thy1 gene in both media alone and ACT-treated samples (Fig. 4B). Furthermore, individual CpG positions within Thy1-CGI1 showed an increase in DNA methylation when treated with the adipogenic cocktail versus media alone, with a significant increase at CpG position 2 (Fig 4C). Methylation decreased across all 5 CpG sites when treated with 5-aza-dC, whether the cells were treated with media alone or with ACT (Fig 4D-E). Four of the five CpG sites examined had significantly reduced methylation levels when cells were treated with ACT and 5-aza-dC. Our data indicates that this region is methylation sensitive and can influence Thy1 expression during adipogenesis.
To test the methylation status of Thy1 over time during adipocyte differentiation, we examined methylation levels at these five sites at days 0 (prior to ACT), 2, 4, and 6. Cells were pretreated with DMSO or given 5-aza-dC for 24 h, then either harvested at day 0 or given ACT medium and with continued treatment with DMSO (vehicle) or 5-aza-dC daily. Overall, average DNA methylation percentages increased in a time-dependent manner when exposed to the cocktail, with the highest degree of methylation occurring on day 6 (Fig 5A). Day 0 samples showed lower methylation levels compared to other time points in which cells had been exposed to ACT. Furthermore, there were no significant changes at any of the 5 CpG sites when given a single dose of 5-aza-dC (Fig. 5B). However, day 6 samples showed a significant two-fold increase in methylation compared to day 0, along with a significant decrease in methylation when cells were exposed daily to 5-aza-dC at all five CpG sites (Fig. 5C). This implies that during the normal adipogenic process, CpG sites at the Thy1 locus become hypermethylated, which may blunt Thy1 expression and allow for differentiation to occur.
Discussion
Excessive adipogenesis can lead to weight gain and obesity, which affects over 700 million people worldwide. The consequences of obesity can be dire, including the development of cardiovascular or liver disease, diabetes, and other comorbidities, which result in significant morbidity and mortality. Therefore, understanding the adipogenic pathway(s) and molecular changes that foster adipocyte differentiation will elucidate the mechanisms contributing to the pathogenesis of obesity. New understanding should lead to better solutions for this growing problem, as lifestyle changes (e.g. improved diet and exercise) are often insufficient. Although genome-wide studies have shown that changes in histone methylation/acetylation occur during adipogenesis25,36,37, few specific adipogenesis-relevant genes have been identified as influenced by epigenetic changes (e.g. DNA methylation). In this study, we identify Thy1 as a methylation sensitive gene and demonstrate that DNA methylation plays an active role in adipogenesis. Ultimately, we found that inhibiting DNA methylation blunts adipogenesis and sustains Thy1 at levels that may retard or suppress adipogenesis.
Thy1 is a cell surface protein that is expressed on mouse thymocytes and on both mouse and human pre-adipocytes38,39. We have previously shown in mouse 3T3-L1 cells that Thy1 is down-regulated in a time dependent manner during adipocyte differentiation, while cells overexpressing Thy1 cells no longer differentiate, even when given an adipogenic cocktail, which typically causes 3T3-L1 cells to differentiate into adipocytes after 6-8 days of exposure. However, here we show that 5-aza-dC blunts adipogenesis, even in the presence of ACT. These findings correlate with reduced levels of Fabp4 expression at both the protein and mRNA levels, along with partially attenuated levels of Thy1 when exposed to a global methylation inhibitor. Fabp4 levels inversely correlate with Thy1 levels; Fabp4 expression increases while Thy1 is suppressed during adipogenesis14. Therefore, blunted levels of Fabp4 are likely another contributing factor that impedes adipogenesis. We also demonstrated that during adipogenesis, Thy1-CGI1 methylation increases, which likely contributes to reduced Thy1 expression. While we saw significant changes at CpG position 2 in ACT-treated samples relative to untreated (media alone) cells (Fig. 4C), we observed similar trends for the other CpG positions. These results are consistent with previous studies showing alterations in methylation status at CpG sites can cause significant changes in gene expression40,41. Treatment with the methylation inhibitor, 5-aza-dC, resulted in hypomethylation of Thy1-CGI1, which in part may contribute to the attenuation of Thy1 expression. Since 5-aza-dC is a global DNA methylation inhibitor, it can affect other genes involved in adipogenesis in addition to Thy1. Our data suggest that DNA methylation is a necessary regulatory mechanism for pre-adipocytes to differentiate into fat cells, consistent with recent reports implying DNA methylation is involved in lineage-specific adipocyte development24,42.
Adipogenesis is a complex process that is controlled by many factors, which include epigenetic and post-transcriptional modifications. Previous studies have established that the expression/activity of microRNAs, small RNAs ~20-22 bp in length that bind to and block the transcription of specific targeted genes, play a role adipogenesis43. Furthermore, in obesity, it has been shown that there are significant changes in the expression of microRNAs (miR), such as an increase in miR-103 levels44,45. We have recently shown that miR-103 levels increase during adipogenesis and can bind the 3’ UTR of Thy1 to blunt its expression15. Here, we confirmed that the levels of miR-103 increase during adipogenesis, and that these levels were unchanged by treatment with 5-aza-dC, suggesting that the regulatory effects of Thy1 methylation are separate from changes in microRNA expression (Figure S3). This represents another potential facet of Thy1 regulation. Therefore, several, possibly overlapping mechanisms are involved in the adipogenic pathway. This has important implications for developing therapeutic interventions to combat adipogenesis and obesity; more than one aspect of this regulatory mechanism may need to be targeted to cause a significant effect.
Stem cells, such as, mesenchymal stem cells (MSCs) are distinguished and defined by various cell surface markers, including Thy146. Mesenchymal stem cells can also differentiate into osteocytes and chondrocytes39, where Thy1 is expressed heterogeneously in each subset47. Recent studies have shown 5-aza-dC prevents adipogenesis and promotes osteoblastogenesis through the activation of Wnt10a30. Wnt10a is known to be upregulated and essential for bone formation48-50. However, Thy1 also plays a critical role51,52. It was recently shown that Thy1 is upregulated during osteoblastogenesis and that Thy1- cells (knockdown and knockout) cannot differentiate into osteoblasts53. However, changes in Thy1 DNA methylation during osteoblast formation have not been analyzed. In our present study, we show that Thy1 levels are sustained, while FABP4 levels are lowered in mMSCs treated with 5-aza-dC in the presence of ACT. While many epigenetic factors aid in stem cell maintenance,54 changes in the methylation status of genes, such as Thy1, may alter stem cell state. Since Thy1 is highly expressed in MSCs and pre-adipocytes, maintaining Thy1 expression may be the key to remaining a precursor cell. However, further investigation is needed. While we saw an increase in Thy1 methylation in mouse pre-adipocyte cells, a crucial next step would be to determine basal Thy1 expression levels and the methylation status of MSCs derived from adipose tissues of obese and non-obese individuals. Testing the ability of these cells to differentiate into fat cells may correlate with their Thy1 expression profiles. Such investigations are likely to provide additional evidence of Thy1’s critical role in adipogenesis and would further underline the importance of our findings.
While we show that 5-aza-dC treatment decreased methylation in the Thy1-CGI1 region and blunted adipogenesis, it is possible that hypomethylating the Thy1 gene at the same time could affect other cell types, such as, fibroblasts (involved in fibrosis); it has been established that Thy1 expression is up-regulated during and involved in myofibroblast differentiation55. Future studies could examine other CpG islands in the traditional promoter region and downstream regions to investigate whether methylation of these sites is also essential for Thy1’s involvement in adipogenesis, fibrosis, and other functions. While the Thy1 gene may be a key target for methylation during adipogenesis, there are undoubtedly other genes regulated by methylation during differentiation. Further investigation is necessary to identify other essential genes, which is fundamental to understanding the adipogenic pathway.
In summary, our work shows for the first time that Thy1 has increased DNA methylation during adipogenesis. We demonstrate herein that inhibiting DNA methylation attenuates the loss of Thy1 when cells are stimulated to differentiate into adipocytes. Blocking methylation leads to sustained Thy1 expression and prevents adipogenesis. These studies further highlight the role of genomic methylation and Thy1’s involvement in adipogenesis, which suggests these pathways may be dysregulated in metabolic diseases in which adipogenesis is elevated, such as obesity.
Materials and Methods
Chemicals
5-aza-2′-deoxycytidine (5-aza-dC), 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and human recombinant insulin were all purchased from Sigma-Aldrich (St. Louis, MO).
Cell Culture
All cells were incubated at 37°C with 7% humidified CO2. 3T3-L1 cells were maintained in 10% calf serum supplemented with DMEM media. C57BL/6 Mouse Bone Marrow Mesenchymal Stem Cells were purchased from Cell Biologics (Chicago, IL) and maintained in 10% mesenchymal stem cell-qualified fetal bovine serum in supplemented MEM media from Thermo Fisher. Cells were plated at 60% confluence and treated at 80% confluency. To induce adipogenesis, media containing an adipogenic cocktail (ACT) was added to confluent cells, which consists of 0.5 mM IBMX, 0.5 μM dexamethasone, and 2 μg/ml insulin. Fresh ACT was added every 2 days. To inhibit methylation, cells were treated daily with 0.5uM 5-aza-dC or DMSO as a control. Cells were then harvested on days indicated per experiment.
Quantitative real-time PCR (qPCR) detection of mRNA
RNA was extracted with a Qiagen miRNeasy Kit and quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE). A BioRad iScript reverse transcription kit was used to make cDNA from 150 ng RNA. RT-qPCR assays were then performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad), according to the manufacturer’s instructions. All genes of interest were normalized to 18S rRNA, and the relative percentages were normalized to 100% to media + DMSO for Thy1 mRNA or 100% to ACT + DMSO for Fabp4 mRNA levels. Primer sequences were as follows Thy1: Fwd 5’-CCTTACCCTAGCCAACTTCAC and Rv 5’-AGGATGTGTTCTGAACCAGC; Fabp4: Fwd 5’-ATGTGTGATGCCTTTGTGGGAAC and Rv5’-TCATGTTGGGCTTGGCCATG; 18s rRNA: Fwd 5’-GTAACCCGTTGAACCCCATT and Rv 5’-CCATCCAATCGGTAGTAGCG.
Western blot analysis
Cells were lysed with 60 mM Tris, 2% SDS, and protease inhibitor cocktail (Sigma-Aldrich). Ten μg of protein was loaded per lane and run on SDS-PAGE gels. Protein gels were transferred to 0.45 um Immobilon-PVDF membranes (Millipore, Temecula, CA) and blocked with 5% BSA in 0.1% Tween 20 in PBS. Primary antibodies, sheep anti-mouse Thy1 (R&D), rabbit anti-mouse Fabp4 (Cell Signaling), and rabbit anti-mouse β-tubulin (Cell Signaling) were diluted 1:5000, 1:500, and 1:5000, respectively, and incubated for 1 h. Membranes were washed in 0.1% Tween 20 in PBS then incubated in anti-sheep or anti-rabbit HRP-conjugated secondary antibodies at 1:5000 or 1:20,000 dilution, respectively. Protein was visualized using Immobilon Western chemiluminescent horseradish peroxidase substrate (Millipore). MagicMark XP protein standard protocol used for ladder (Novex). Blots were developed by X-ray film. All blots are provided as uncropped images in the supplementary data.
Flow cytometry
Cells were trypsinized and washed in PBS, then fixed with 2% PFA and blocked with 1:50 human Fc receptor blocker (Miltenyi Biotech Inc., San Diego, CA) in PBS. The cells were then incubated with anti-mouse Thy1.2-PE conjugated antibody, 1:500, (BD Biosciences, San Jose, CA) for 1 h on ice. Cells were washed and resuspended in PBS. Cells were analyzed on a LSR II flow cytometer running FACSDIVA software (BD Biosciences). Analysis of fluorescence data was performed using FlowJo software v10.1 (FlowJo, LLC, Ashland, Oregon).
Immunofluorescent staining
Cells treated in 12-well plates were washed with 1X PBS and fixed with 2% PFA for 10 min and washed three times with PBS. Cells were blocked in 1% BSA and 0.1% Triton X-100 in PBS with normal donkey serum (Jackson Immunoresearch) and Fc-blocker 1:50 (BD Biosciences). The primary antibodies used were Thy1.2-PE conjugated antibody, (BD Biosciences, San Jose, CA) and Fabp4 (Cell Signaling), which were diluted 1:500 in 1% BSA and incubated for 2 h at room temperature in the dark. After removal of primary antibody and three washes, secondary antibody (donkey anti-rabbit AF647) was applied at a 1:2000 dilution for an hour. Cells were then washed and visualized on an EVOS-FL Cell Imaging System (Thermo Fisher).
DNA extraction and Bisulfite conversion
Genomic DNA was isolated from cells using a DNeasy DNA extraction kit (Qiagen, Valencia, CA) and quantified using the NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE). 1000 ng of genomic DNA was then bisulfite converted using an Epitect Plus Bisulfite Conversion Kit (Qiagen) to be analyzed by a pyrosequencer.
Pyrosequencing assays
Bisulfite-treated DNA was amplified using the PyroMark PCR kit (Qiagen, Valencia, CA) with the conditions of, 95°C for 5 min, 45 cycles of (95°C for 30 s, annealing temperature of 58°C for 30 s, 72°C for 30 s), 72°C for 10 min. The PCR product sizes were then verified with electrophoresis on a 2% agarose gel. Ten µl of the biotinylated PCR products were mixed with 1 μl streptavidin-coated Sepharose beads, 40 μl PyroMark binding buffer (Qiagen), and 29 μl RNAase-free water for a total volume of 80 μl. This mixture was then run on a PyroMark Vacuum Workstation (Qiagen). The purified PCR products were then added to the annealing buffer, which contained the corresponding sequencing primer. After annealing, the plate was loaded into the PyroMark Q96 MD instrument (Qiagen). PyroMark-CpG software automatically generates a dispensation order of dNTPs and control dispensations, based on the sequence to analyze. Controls are included in the dispensation order to check the performance of the reactions. All runs also included a no template control. We analyzed the data with the PyroMark software for quantification of % DNA CpG methylation.
Pyrosequencing Primers
Methylation levels were measured in the first CpG island of intron 1, which is part of the promoter17,20-23 of mouse Thy1 (chr9:44,043,384-44,048,579; GRCm38/mm10) (94bp-349bp) using the pyrosequencing assay. Gene-specific primers for Thy1 were designed using the Pyro-Mark assay design software, version 2.0 (Qiagen, Valencia, CA). The program automatically generated primer sets that included both PCR and sequencing primers, based on selected target sequences. One of the primers was biotinylated to enable immobilization to streptavidin-coated beads. The sequences were as follows: Forward primer: 5’-TTTAGTTATAGTTTTGGGAAAGGATAT Reverse Biotinylated primer: 5’-CCACCTCCTCCCTCTATT Sequencing primer: 5’-ATAGGGAFTTTTTATAT
Statistical analysis
All values are presented as mean ± SEM. Experiments were conducted in triplicate at separate times. Two-way analysis of variance (ANOVA) were used for statistical analysis using GraphPad Prism6. P-values < 0.05 were considered significant.
Authors Contributions
E.F. performed all experiments. E.F. C.W. M.S. R.P. aided in experimental design and data analysis. E.F. prepared figures and wrote main manuscript. All authors aided in editing the manuscript. All authors have reviewed the manuscript.
Additional Information
Competing Interests: The authors declare no competing interests.
Acknowledgements
This project is funded by NIH grants F31ES027767, TL1-TR000096, ES001247, and ES0023032.
Footnotes
The authors have no conflict of interest.
Work funded by F31ES027767, TL1-TR000096, ES001247, and ES0023032