Cell culture and RNA extraction

Skeletal muscles from Korean native cattle aged 22-24 months with an average body weight of 550-600 kg were used for this experiment. All animals were handled according to a protocol approved by the Animal Care and Concern Committee of the National Institute of Animal Science, Korea. Briefly, the collected muscle was minced into fine pieces and digested with trypsin-EDTA (GIBCO, CA, USA), after which the samples were centrifuged at 90×g for 3 min and the upper phase was passed through a 40-μm cell strainer. The filtrate was then centrifuged at 2,500 rpm, after which the cell pellet was collected, washed twice and cultured in Dulbecco’s modified Eagle’s medium (DMEM; HyClone Laboratories, UT, USA) supplemented with 10% fetal bovine serum (HyClone Laboratories) and 1% penicillin/streptomycin at 37°C under 5% CO₂. The emphasis was given on the primary MSC condition and the culture medium was changed every other day. Cells were treated with a transdifferentiation cocktail (TDC) at 70% confluency and allowed to grow for 7 days. To induce differentiation, cells were allowed to grow in DMEM without reducing serum (DMEM with 10% FBS and 1% P/S). A detailed description of MSCs isolation, differentiation, and transdifferentiation is described in our previous studies [6,21]. Cells were harvested for RNA isolation on day 10 of culture for MSCs and day 14 for MFCs. Similarly, MSCs switched to transdifferentiation media on day 10 were harvested on day 17 for ALC. Total RNA extraction and mRNA purification were conducted according to Lee et al. [11]. Briefly, equal amounts of RNA samples collected from cultured MSCs, MFCs and ALCs were pooled together, after which mRNA was purified from each pool of total RNA (100 μg) using the absolutely mRNA purification kit (Stratagene, CA, USA) and used for subsequent library construction. As for C2C12 cells, the cells cultured in 10% FBS media were switched to 2% FBS differentiation media when they reached 70% confluence. The cells were then harvested in Trizol, after which RNA was isolated and stored in DEPC water at -80°C until use. Total RNA extraction and cDNA synthesis were conducted as previously described [6].

Normalized cDNA library construction

Normalized cDNA libraries from bovine primary cells were generated using the duplex-specific nuclease (DSN) based normalization method described by the manufacturer (Evrogen JSC, Russia). A directional Lambda ZAP cDNA Synthesis and Gigapack III Gold Cloning Kit (Stratagene, CA, USA) was used for construction of the cDNA library. Briefly, 5 μg of mRNA was reverse transcribed for first strand cDNA synthesis using an oligo-dT linker-primer containing a XhoI cloning site by incubating the samples at 42°C for 1 hour. To synthesize the second cDNA strand, RNase H (1.5 U/ μl) and DNA polymerase-I (9.0 U/μl) enzymes were added and synthesized for 2.5 hrs at 16°C. EcoRI linkers were then ligated into the 5´-termini of cDNA, after which normalization of the cDNA library was conducted using DSN as previously described [13].The products were then separated on a column containing Sepharose® CL-2B gel filtration medium to yield three fractions ranging in size from 500 bp to 1.5 kb, which were subsequently ligated into ZAP Express vector (pBK-CMV) to produce primary libraries of MSCs, MFCs, and ALCs. In vitro packaging of the ligation product was conducted using a ZAP Express cDNA Gigapack III Gold Cloning Kit (Stratagene, CA, USA). Excision cloned fragments were packed in phagemids and infected with E.coli strain XLOLR. The E. coli were then plated on LB-Kanamycin (50 μg/ml) containing X-gal/IPTG for blue/white selection. White colonies were randomly and manually selected and inoculated into a 384-well plate (Corning, NY, USA) containing 40 μl TB with Kanamycin (50 μg/ml), after which the samples were then incubated for 16 hours at 37°C, mixed with glycerol solution (65% glycerin, 0.1M MgSO₄, 0.025M Tris-HCl, pH 8.0) and stored at -80 °C. Insert sizes were confirmed by PCR.

DNA sequencing

Sequencing of bovine MSCs, MFCs, and ALCs cDNA clones was performed as previously described [13]. Briefly, single plasmid colonies were cultured in terrific broth (TB) medium supplemented with Kanamycin (50 μg/ml) and then plasmid was purified using alkaline lysis method [22,23]. Sequencing reactions were performed using 250 ng plasmid DNA as a template with the3 pmol T3 primer (5'-ATTAACCCTCACTAAAG-3') and Big Dye Terminator v3.1 using a GeneAmp PCR System 9700 (Applied Biosystems, CA, USA). PCR products were purified by ethanol precipitation and DNA sequences were obtained using an ABI 3730 XL DNA Analyzer (Applied Biosystems). The nucleotide sequences obtained in this study have been submitted and are available in DNA Data Bank of Japan (DDBJ) under accession number HX915285- HX939203.

EST analysis

The Phred software [24,25] was used for base calling and quality assignment of the chromatogram files obtained from the sequencer. The trace files were trimmed using trim-alt 0.05 (Phred score ≥ 20). Cross-match software was used to identify and trim off each vector sequence, and EST sequences shorter than 100 bp were discarded. The TGICL [26] package and cap3 software [27] were used to assemble and cluster ESTs. Finally, the clusters and singletons were analyzed by means of a homology based search using a local BLAST [28] against the NCBI non-redundant (nr) database, NLM, USA. Significant matches were determined when the expected value was <1 x e^-5 (Figure 1).

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Figure 1. Schematic diagram for EST data analysis.

https://doi.org/10.1371/journal.pone.0079780.g001

Functional studies and pathway analysis

The function of ESTs was predicted through KOG (clusters of orthologous groups for eukaryotic complete genomes) analysis [29]. A stand-alone BLAST system (http://www.ncbi.nlm.nih.gov/COG/grace/kognitor.html) was used for functional classification of the reference sequences. Differentially expressed ESTs were categorized into 25 functional groups by the KOG database and the BLASTX program in NCBI (<1 × e^-10) [29].

DAVID [http://david.abcc.ncifcrf.gov/home.jsp] functional annotation cluster analysis was performed to measure enrichment of the gene ontology (GO) terms within these clusters in MSC, MFC and ALC. Only genes for which at least 5 ESTs were detected were selected from the EST data set, while genes with less than 5 ESTs were excluded from analysis. Additionally, GO terms that reported a p-value of ≤ 0.05 and number of genes ≥ 5 were selected. The GO terms of cellular components, molecular function and biological processes in DAVID were employed to categorize enriched biological categories in three gene lists.

Pathway mapping for MSC, MFC and ALC was performed using the KEGG Automatic Annotation Server (KAAS) [30]. KAAS offers functional annotation of genes in a genome via a BLAST similarity search against a manually curated set of ortholog groups in the KEGG GENES database. KAAS assigned a KEGG Orthology (KO) number to genes in the data sets, which were mapped to one of KEGG’S reference pathways.

Identification of MSC, MFC and ALC specific genes

To identify MSC, MFC and ALC specific genes, only genes having at least 5 ESTs in one category and none in the other two categories were selected. For example, MSC specific genes were those with at least 5 ESTs in the MSC category but 0 ESTs in MFC and ALC.

Real time RT-PCR analysis

Genes with high EST numbers were further studied in C2C12 cells and their expression was confirmed by real time RT-PCR. Briefly, cDNA was synthesized from RNA using Superscript-II reverse transcriptase (Invitrogen). A total of 1μg of RNA (20 µl total volume) was primed with oligo (dT)₂₀ primers (Bioneer, Daejeon, Korea), and reverse transcription was then carried out in a thermal cycler by subjecting the samples to 42°C for 50 min and 72°C for 15 min. PCR was subsequently conducted using 2 µl of the 5× diluted cDNA product and 10 pmoles of each gene-specific primer using a 7500 real-time PCR system (Applied Biosystems, CA, USA). Power SYBR® Green PCR Master Mix (Applied Biosystems) was used as the fluorescence source. All primers used were designed with the Primer 3 software (http://frodo.wi.mit.edu) using sequence information listed at the National Center for Biotechnology Information (primer information is provided in Table S1).

Oil red O staining

Oil red O working solution was prepared asa6:4 dilution of a stock solution (3.5mg/ml Oil red O powder in 100%isopropanol) in distilled water and filtered through Whatman filter paper No. 2. Briefly, 10% formaldehyde-fixed, PBS washed cells were incubated for 1 hr with 1 ml of Oil red O working solution. The cells were then washed with 60% isopropanol and PBS, respectively, after which pictures were taken using a light microscope equipped with a digital camera (Nikon, Tokyo, Japan). To quantify Oil red O in cells, the plates were dried and 500 µl of 100% isopropanol was added to elute the color. The optical density was then measured at 510 nm using a Versa Max microplate reader (Molecular Devices, CA, USA).

Immunocytochemistry

C2C12 cells in a covered glass-bottom dish were treated with differentiation/transdifferentiation medium and stained for DDAH2 or HBA2 antibody. DDAH2 was stained after 48 hrs and HBA2 was stained after 24 hrs. Bovine MSCs in a covered glass-bottom were cultured at Day 11 and stained with MyoD antibody. Briefly, cells were rinsed with PBS, fixed in 4% formaldehyde, permeabilized by 0.2% TritonX-100,after which the signals were enhanced using an Image-iT^TM FX signal enhancer (Invitrogen, CA, USA). The cells were then incubated with rabbit primary antibody (DDAH2/HBA2/MyoD, Santa Cruz Biotechnology, CA, USA) at 4°C in a humid environment overnight. Secondary antibody (Alexa Fluor 488 goat anti-rabbit SFX kit; Molecular Probes, Eugene, OR, USA) was treated for 1 hr at room temperature followed by nuclear stainingwith4´,6´-diamino-2-phenylindole (DAPI; Sigma-Aldrich, MO, USA). Pictures were taken using a fluorescent microscope equipped with a digital camera (Nikon).

Western blot

Total protein was isolated from cells treated with differentiation or transdifferentiation media and cultured for different lengths of time. Briefly, cells washed with ice-cold PBS and lysed in RIPA lysis buffer with protease inhibitor cocktail (Thermo Scientific, FL, USA) were used for Western blot analysis. Total protein was quantified by the Bradford method using protein assay dye solution [31]. Briefly, 50 µg of protein were electrophoresed in 10% SDS-polyacrylamide gel after reducing at 90°C for 3 min with β-mercaptoethanol and then transferred to a PVDF membrane. Membranes were blocked and hybridized with DDAH2 (1:500), HBA2 (1:500), or ß-actin antibody (1:2000) (Santa Cruz Biotechnology, TX, USA) overnight at 4°C. Blots washed in TBST were then incubated with horseradish peroxidase conjugated secondary antibody for an hour at room temperature. Finally, the blots were developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, USA).

Immunohistochemistry

HBA2 and DDAH2 expression in bovine tissues was evaluated by immunohistochemistry. Briefly, paraffin-embedded tissue sections were deparaffinized, hydrated, and then quenched for endogenous peroxidase activity in 3% H₂O₂ for 15 min. The sections blocked with 5% goat serum were subsequently incubated with either HBA2 or DDAH2 antibody (2 μg/mL, Santa Cruz Biotechnology) overnight at 4°C. Next, the sections were washed in PBS three times, after which they were incubated with goat anti-rabbit IgG HRP (Santa Cruz Biotechnology) for 1 hr at RT. Positive signals were visualized by adding diaminobenzidine and hydrogen peroxide as substrates. A negative control experiment was also carried out by omitting the primary antibody. Stained sections were counterstained with hematoxyline, washed in running tap water, and then dehydrated, mounted, and examined using a light microscope.

Knockdown of DDAH2 and HBA2

Transfection of C2C12 cells by siRNA was conducted with either 150 nM of HBA2-siRNA or 50 nM of DDAH2-siRNA (Dharmacon) complexed with 5µl of lipofectamine (Invitrogen) in Optimem (Gibco, NY, USA). Cells were allowed to recover and grow in culture media (DMEM+ 10% FBS+ 1% P/S) until they reached 80% confluence, after which cells were treated in differentiation media (DMEM with 2% FBS and 1% P/S) for DDAH2 and transdifferentiation media for HBA2and grown to the designated time-point.

Statistical analysis

The normalized mean expression was compared using Tukey’s Studentized Range (HSD) to identify significant differences in gene expression. A nominal p-value of less than 0.05 was considered to be statistically significant. Real time RT-PCR data were analyzed by one-way ANOVA using PROC GLM in SAS package ver. 9.0 (SAS Institute, NC, USA).

Normalized cDNA libraries and EST analyses

MSCs, MFCs and ALCs cultured from bovine hind leg muscle were used to construct three normalized cDNA libraries. Both the MFCs and ALCs exhibited prominent myotube formation and intracellular lipid accumulation following differentiation and transdifferentiation respectively (Figure S1). During this procedure, we determined the purity of the isolated cells by single cell culture and found that at least 83% were MSCs (data not shown). Nuclear localization of MyoD expression was evident in majority of MSCs when analyzed by immunocytochemistry (Figure S2). Thus, the three normalized cDNA libraries were successfully constructed from MSCs, MFCs, and ALCs. Titration of the libraries resulted in 1.4x10⁶, 5x10⁵, and 3x10⁶ independent clones for MSCs, MFCs, and ALCs, respectively. A total of 24,192 clones (8,064 clones from each library) were randomly selected for DNA sequencing. Vector trimming and elimination of lower quality sequences resulted in a total of 23,919 ESTs that included 7,974 from MSCs, 7,991 from MFCs and, 7,954 from ALCs. Comparison of the success rate of sequencing among these libraries with clusters, singletons, and contigs is shown in Table 1. The average length (bp) of ESTs was 788, 792, and 776 for MSCs, MFCs, and ALCs, respectively. ESTs assembled and clustered for all the three libraries resulted in 3,333 clusters, 5,517 singletons and 3,842 contigs.

Gene expression in MSCs, MFCs, and ALCs

The ESTs obtained during sequencing were annotated by a BLAST search of the NCBI database. Overall, 20,266 ESTs showed known annotation and 2,526 (940, 686 and 900 for MSC, MFC, and ALC groups, respectively) were unannotated. Gene annotation of the identified ESTs with higher numbers during MSCs, MFCs, and ALCs formation is shown in Table 2. Among these genes, 37% were also observed by microarray analysis. Genes known to be involved in myogenesis, myosin regulatory light chain 2 (MYL2), skeletal muscle isoform (MYLPF) and actin, alpha skeletal muscle (ACTA1), were only identified during MSC and MFC formation. Similarly, S100 calcium-binding protein A4 (S100A4) and DDAH2 which have no previous reported role during myogenesis were identified with higher EST numbers during MFC formation. Additionally, pentraxin related protein 3 (PTX3), lysophospholipid acyltransferase (LPCAT4) and HBA2 were identified with high EST numbers during ALC formation. Almost equal expression of fibronectin 1 (FN1) was observed in both MFCs and ALCs.

Functional study and pathway analysis of ESTs

The differentially expressed genes identified by ESTs were functionally categorized by querying the NCBI eukaryotic Orthologous Group (KOG) database. Twenty-five different functional classes were formulated and summarized into four functional groups, information storage and processing, cellular processes and signaling, metabolism, and poorly characterized. A total of 16,048 ESTs (MSCs=5,534, MFCs=5,265, ALCs=5,249) were analyzed using the KOG database, among which the highest percentage was related to cellular processes and signaling. Moreover, genes related to translation, ribosomal structure and biogenesis, posttranslational modification, protein turnover, chaperones and energy production and conversion were enriched during MSCs, MFCs and ALCs formation (Figure 2). A large number of ESTs represented genes related to signal transduction, cytoskeletons and extracellular structures (Table 3A) during MFC formation. Latent transforming growth factor beta binding protein 2 (LTBP2), tubulin alpha-1B chain (TUBA1B) and 40S ribosomal protein SA (RPSA) were found to be the genes with the highest ESTs in these categories. Similarly, during ALC formation, ESTs related to lipidtransport and metabolism, carbohydrate transport and metabolism and energy production and metabolism were abundant (Table 3B). Fatty acid binding protein 4 (FABP4), 2-oxoglutarate dehydrogenase and HBA2 showed the highest EST numbers. In contrast, transgelin (TAGLN), osteonectin (ON) and cytoskeletal beta actin showed almost equal numbers of ESTs in MSCs, MFCs and ALCs (data not shown), suggesting their equal contribution during MSC differentiation and transdifferentiation.

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Figure 2. KOG analysis of ESTs in MSC, MFC and ALC.

The functions of genes were categorized and each function is represented by the symbols given below: .

[J] Translation, ribosomal structure and biogenesis, [A] RNA processing and modification, [L] replication, recombination and repair, [B] chromatin structure and dynamics, [Y] nuclear structure, [V] defense mechanisms, [T] signal transduction mechanisms, [M] cell wall/membrane/envelope biogenesis, [N] cell motility, [Z] cytoskeleton, [W] extracellular structures, [U] intracellular trafficking, secretion, and vesicular transport, [O] posttranslational modification, protein turnover, chaperones, [C] energy production and conversion, [G] carbohydrate transport and metabolism, [E] amino acid transport and metabolism, [F] nucleotide transport and metabolism, [H] coenzyme transport and metabolism, [I] lipid transport and metabolism, [P] inorganic ion transport and metabolism, [R] general function prediction only, [S] function unknown. The X-axis represents percentage and Y axis represents functional category.

https://doi.org/10.1371/journal.pone.0079780.g002

To classify biological processes coordinately regulated during MSC, MFC and ALC formation, we further created three subsets of all highly expressed genes by keeping those genes for which the number of ESTs was ≥ 5. All other genes with < 5 ESTs were discarded. These highly expressed subsets consisted of 233 MSC (MSC233), 258MFC (MFC258), and 248ALC (ALC248) genes, respectively. These three highly expressed gene lists were then used for functional annotation by employing the Functional Annotation Cluster (FAC) tool available in the Database for Annotation, Visualization, and Integrated Discovery (DAVID) [http://david.abcc.ncifcrf.gov/home.jsp]. Table S2 provides the list of these highly expressed subsets and the genes annotated by DAVID. The GO terms “Biological Process,” “Cellular Component” and “Molecular Function” were used for annotations. DAVID FAC analysis of MFC258 produced a total of 41 functional clusters using default parameters. Similarly, FAC analysis of MSC233 genes resulted in 22 clusters, whereas 31 clusters were reported for ALC248 genes. GO terms having at least ten genes from the resulting functional analysis with statistically significant p-values for these three subsets are listed in Table 4 and a complete list is available in Table S3. Among these categories, genes involved in the extracellular region, extracellular matrix (ECM), structural molecule activity, non-membrane-bounded organelle, cytoskeleton, calcium ion binding, ribonucleoprotein complex and various carbohydrate metabolic processes were the most represented groups, indicating that the cells were undergoing rapid structural rearrangement for cellular differentiation. High enrichment of GO terms such as ECM, cytoskeleton, and carbohydrate metabolism were found in all the three sets. However, some specific terms for each category were also identified. MFC showed a firm preference for terms that involve adhesion, endopeptidase activity and embryonic development. Similarly, MSC includes processes such as cellular homeostasis, oxidative phosphorylation, as well as myofibril and contractile fiber, whereas ALC had a strong inclination toward phosphate metabolic processes and kinase activity. Commencement of differentiation in cells leads to intense alterations in the transduction of locomotion and cell shape controlling proteins and depends on reorganization of their cytoskeleton and plasma membranes [32].

We also identified the biochemical pathways of MFC258, MSC233 and ALC248 genes annotated in the present study. FASTA formatted amino acid sequences of DAVID annotated genes in these sets were fed into the KAAS for prediction of various pathways. A total of 130 pathways were predicted for MFC258, whereas 114 and 128 pathways were predicted for MSC233 and ALC248, respectively. A representative pathway for each of the three categories is shown in Figure 3 and a complete list of all pathways is provided in Table S4. Proteins involved in various stages of cellular differentiation pathways including proteoglycans in cancer, regulation of actin cytoskeleton, focal adhesion, tight junction, ribosome, oxidative phosphorylation, ECM-receptor interaction, and various important signaling pathways including the MAPK signaling, Wnt signaling, Hippo signaling, TGF-beta signaling, PI3K-Akt signaling and calcium signaling pathways were represented by unigenes derived from our EST dataset. These data provide substantial evidence that the ESTs generated in this study offer an important resource for MSC differentiation related gene discovery and future functional studies.

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Figure 3. Biochemical pathway analysis of highly expressed gene list.

Representative KEGG pathway for A) MSC, B) MFC and C) ALC.

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Identification of MSC, MFC and ALC specific genes

We also attempted to determine if our EST dataset represented MSC, MFC and ALC specific genes (e.g., genes that were detected in MFC only and absent from MSC and ALC). Based on the criteria mentioned in the methods section, we identified 25, 28 and 36 genes that were present in MSC, MFC and ALC only (Table 5). From this table, extremely high expression of PTX3, HBA and LPCAT4 was observed in ALC only. Similarly, adapter-related protein complex 3 mu-1 subunit (AP3M1) and 60S ribosomal protein L3 (RPL3) were among a few genes expressed in MFC and MSC, respectively. Over-representation of these MSC, MFC and ALC specific genes (esp. PTX3 and HBA) reflects their distinctiveness and indicates that they may serve as potential candidates for future studies conducted to elucidate their role in muscle cell differentiation.

mRNA and protein expression

Expression of identified genes that showed high EST numbers was validated by real time RT-PCR. mRNA expression was checked at different time points during differentiation and transdifferentiation of C2C12 cells. DDAH2 showed more than 4 fold induction at Day 2 when compared to Day 0, while S100A4 maintained its pattern of expression from Day 1 to 3 during MFC formation (Figure 4A). PTX3 mRNA expression reached as high as 12 fold on Day 2, whereas HBA2 reached almost 5 fold at Day 1during ALC formation (Figure 4B). Protein expression of the two representative genes was confirmed by immunoblot and its cellular localization was visualized by immunocytochemistry in C2C12 cells. A time dependent increase in the concentration of protein was observed by Western blot (Figure 4C) and increased cytoplasmic expression of DDAH2 protein was evident upon immunocytochemistry (Figure 4D). Similarly, expression of HBA2 was detected on transdifferentiated C2C12 cells from as early as day 1, and gradually increased to day 3 (Figure 4E). Moreover, clear localization of cytoplasmic HBA2 protein was evident at day 2 upon immunocytochemistry analysis (Figure 4F). Immunohistochemistry of DDAH2 and HBA2 on tissue sections from bovine skeletal muscle revealed in vivo muscle expression of these proteins (Figure 4G and H).

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Figure 4. mRNA expression of genes identified with higher EST numbers.

C2C12 cells treated with differentiation and transdifferentiation media at 80% confluence were harvested at different time points. Real time PCR was carried out with cDNA synthesized from1µg of total RNA. mRNA expression analysis of most of the genes showed more than 2 fold induction during MFCs (A) and ALCs (B) formation, respectively. Western blot analysis of DDAH2 shows a gradual increase in protein expression with time (C). Cellular localization of DDAH2 by immunocytochemistry during myogenesis. The first column shows cell pictures at Day 0 and Day 3. The second column shows expression of DDAH2 and the third column shows a merged image of DAPI-stained nuclei and DDAH2 IF (D).Western blot analysis of HBA2 expression during transdifferentiation confirms its protein level expression (E). Cytoplasmic localization of HBA2 by immunocytochemistry during transdifferentiation. The first column shows cell pictures at Day 0 and Day 2, the second column shows expression of HBA2 IF and the third column shows a merged image of DAPI-stained nuclei and HBA2 IF (F). DDAH2 and HBA2 immunohistochemistry of bovine skeletal muscle (G and H). Day 0 represents control (mean ± S.D., n= 3). p-value indicates the statistical significance of the data and different letters show significant differences among groups.

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Knockdown of DDAH2 and HBA2

To confirm the role of genes identified during differentiation and transdifferentiation, gene knockdown by siRNA was employed in DDAH2and HBA2. DDAH2_kd by siRNA showed a 40% reduction in mRNA expression with a slight change in cell morphology (Figure 5A&B). Interestingly, DDAH2_kd showed a decrease in MYOG upto 50% during myogenesis (Figure 5A). Furthermore, HBA2 silencing in C2C12 cells showed an upto 70% reduction in its expression during transdifferentiation (Figure 6A). CD36, which is a marker gene for adipogenesis, was checked in HBA2_kd and its expression was found to be reduced up to 50%, whereas FABP4 was slightly decreased (Figure 6 B). The effects of HBA2_kd on intracellular lipid formation followed by its treatment with transdifferentiation media were also evaluated at day 5 by Oil Red O (O-R-O) staining and the results showed a 20% reduction in intracellular fat during ALC formation (Figure 6C& D).

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Figure 5. DDAH2 knockdown in C2C12 cells during myogenesis.

mRNA expression of DDAH2 and MYOG after DDAH2_kd during differentiation in C2C12 at Day 2 (A). Representative cell picture showing morphological changes in DDAH2_kd cells (B). Mock represents control (mean ± S.D., n= 3). p-value indicates the statistical significance of the data and different letters show significant differences among groups.

https://doi.org/10.1371/journal.pone.0079780.g005

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Figure 6. HBA2 knockdown in C2C12 cells during transdifferentiation.

(A) mRNA expression of HBA2 after HBA2_kd during transdifferentiation in C2C12 at Day 1, and (B) HBA2_kd effect on mRNA expression of different adipogenic marker genes.(C) Cell picture following intracellular lipid staining by O-R-O on Day 5 during transdifferentiation in HBA2_kd and Mock cells. Quantification of O-R-O at 510 nm. Control represents Mock (mean ± S.D., n= 3). p-value indicates the statistical significance of the data and different letters show significant differences among groups.

https://doi.org/10.1371/journal.pone.0079780.g006