Cellular Signalling 26 (2014) 2107–2116

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Mechanism of SB431542 in inhibiting mouse embryonic stem cell differentiation Juan Du a,c, Yongyan Wu b,c, Zhiying Ai a,c, Xiaoyan Shi a,c, Linlin Chen a,c, Zekun Guo b,c,⁎ a b c

College of Life Sciences, Northwest A&F University, 3 Taicheng Road, Yangling 712100, Shaanxi, China College of Veterinary Medicine, Northwest A&F University, 3 Taicheng Road, Yangling 712100, Shaanxi, China Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, 3 Taicheng Road, Yangling 712100, Shaanxi, China

a r t i c l e

i n f o

Article history: Received 10 April 2014 Received in revised form 9 June 2014 Accepted 9 June 2014 Available online 18 June 2014 Keywords: SB431542 Undifferentiated state Smad2/3 Differentiation

a b s t r a c t SB431542 (SB) is an established small molecular inhibitor that specifically binds to the ATP binding domains of the activin receptor-like kinase receptors, ALK5, ALK4 and ALK7, and thus specifically inhibits Smad2/3 activation and blocks TGF-β signal transduction. SB maintains the undifferentiated state of mouse embryonic stem cells. However, the way of SB in maintaining the undifferentiated state of mouse embryonic stem cells remains unclear. Considering that SB could not maintain embryonic stem cells pluripotency when leukemia inhibitory factor was withdrawn, we sought to identify the mechanism of SB on pluripotent maintenance. Transcripts regulated by SB, including message RNAs and small non-coding RNAs were examined through microarray and deep-sequence experiments. After examination, Western blot analysis, and quantitative real-time PCR verification, we found that SB regulated the transcript expressions related to self-renewal and differentiation. SB mainly functioned by inhibiting differentiation. The key pluripotent factors expression were not significantly affected by SB, and intrinsic differentiation-related transcripts including fibroblast growth factor family members, were significantly down-regulated by SB. Moreover, SB could partially inhibit the retinoic acid response to neuronal differentiation of mouse embryonic stem cells. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Embryonic stem cells (ESCs) isolated from the inner cell mass of the blastocyst can self-renew indefinitely or differentiate into all cell types under defined conditions [1]. These ESC characteristics are an excellent material for the investigation of the ESCs self-renewal regulation mechanism, particularly cell type-commitment methods, and treatments using ESCs for disease therapies [2–5]. Maintaining the indefinite proliferation and undifferentiated state of ESCs in vitro is difficult because ESCs should be cultured on feeder cell-coated plates supplied with additional supporting factors, such as leukemia inhibitory factor (LIF), bone morphogenetic protein 4 (BMP4), and basic fibroblast growth factor (FGF) [6,7]. The method of culturing ESCs in feeder cells and exogenous factor-containing conditions complicates the ESCs background. The feeder cells used for ESCs culture limit the ESCs clinical application because of possible contamination. The addition of the exogenous factor to feeder cell-supported ESCs causes difficulty in determining whether the exogenous factor acts directly on ESCs or through the feeder cells. Therefore, simpler and easier ESC culture methods are needed. ⁎ Corresponding author at: College of Veterinary Medicine, Northwest A&F University; Key Laboratory of Animal Biotechnology, Ministry of Agriculture, 3 Taicheng Road, Yangling 712100, China. Tel./fax: +86 29 87080092. E-mail address: [email protected] (Z. Guo).

http://dx.doi.org/10.1016/j.cellsig.2014.06.002 0898-6568/© 2014 Elsevier Inc. All rights reserved.

Recently, small molecules have been reported to manipulate the ESCs fate under feeder- and/or serum-free conditions. SC1 (also known as pluripotin) can directly bind and inhibit the RasGAP and ERK1/2 activation and thus maintain the self-renewal and pluripotent state of mouse ESCs (mESCs) under defined chemical conditions without feeder cells, LIF, and serum [8]. BIO, a specific GSK3 inhibitor, can indirectly activate the Wnt/β-catenin pathway and further maintain the undifferentiated state of ESCs under feeder-free conditions [9]. PD0325901 and CHIR99021 are two small molecules that inhibit MEK and GSK3, respectively, and they maintain the ESCs self-renewal under LIF- and serum-free conditions [10]. Small molecules, which can directly participate in a specific signaling pathway and can be easily withdrawn from the cell culture medium, have been considered a favorable tool in revealing the determination mechanisms of the ESCs fate and the method to control ESCs growth. SB431542 (5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2yl)-benzamide, indicated as SB thereafter) is a competitive ATPbinding site inhibitor of activin receptor-like kinase 4 (ALK4), ALK5, and ALK7, which are TGF-β type 1 receptors that specifically block Smad2/3 mediated-signaling transduction [11]. SB induces the differentiation of human ESCs (hESCs) [12,13], and maintains the undifferentiated state of mESCs [14]. Moreover, SB combined with the other inducer factors (i.e., Klf4, Oct4, and c-Myc/Sox2) can replace Sox2 and c-Myc to induce the pluripotent stem cells from fibroblast cells [15].

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Currently, many genes regulated by SB have been discovered, and pathways including Bmp4-Smad1/5/8-Ids have been shown to be influenced by SB [16]. However, the influences of SB on mESCs pluripotency maintenance remain ambiguous. To know further how SB maintains mESCs pluritotency, we treated the feeder-free cultured J1 mESCs with SB and performed microarray and deep-sequence experiments to explore the mechanisms of SB in maintaining the undifferentiated state of ESCs. On the basis of the morphological changes in J1 mESCs cultured in defined SB conditions, microarray, and deep-sequence data (confirmed by Western blot analysis and quantitative real-time PCR (qPCR) examination), we found that SB primarily maintains the undifferentiated states of ESCs by inhibiting differentiation. 2. Materials and methods 2.1. Reagents Unless otherwise indicated, reagents used in this study were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Rabbit antiNanog polyclonal antibody was obtained from Bethyl Laboratories (Montgomery, TX, USA). Rabbit anti-Smad1, rabbit anti-phosphoSmad1/5, rabbit anti-phospho-Smad2, and mouse anti-Smad2 were purchased from Cell Signaling Technology (Beverly, MA, USA). Mouse anti-Gapdh and mouse anti-c-Myc antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Mouse anti-HA, mouse anti-Myc, mouse anti-Flag, and all of the second antibodies used in this study were purchased from Beyotime Institute of Biotechnology (Beyotime, Jiangsu, China). 2.2. Cell culture J1 mESCs purchased from ATCC (Manassas, VA, USA) were maintained in 0.1% gelatin-coated plates (Nunclon, Roskilde, Denmark) with Dulbecco's modified Eagle's medium containing KnockOut™ serum replacement (15%), non-essential amino acids (0.1 mM), Lglutamine (2 mM), penicillin (125 μg/mL), streptomycin (100 μg/mL), β-mercaptoethanol (0.1 mM), and 1000 U/mL LIF (ESGRO, Millipore, USA). All other reagents used for cell culture were purchased from Gibco (Invitrogen, Carlsbad, CA, USA).

absent—A in the data) in the control and SB-treated samples, the obtained microarray data were annotated using an SAS analysis system. Gene expression changes were characterized as fold change (FC), which compared the gene expression levels between the SB- and DMSOtreated J1 mESCs. The original microarray data can be accessed in the NCBI GEO database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi? acc=GSE55729). For the whole-genome miRNAs expression, the quality of Trizolisolated total RNAs was examined and the sequence was analyzed by the Beijing Genomics Institute (Shenzhen, Guangdong, China). In brief, the total RNA extracted was separated with PAGE and then purified for the small RNA molecules (less than 30 bases). The purified small RNA molecules were ligated using a Solexa adaptorer, amplified with a PCR, and analyzed using their sequences. After filtering the adapter sequences and contaminated reads, the retained sequences were processed for normalization and annotation. The exchanges of miRNAs expression were presented as FC, which was calculated as the normalized expression of the treatment divided by the normalized expression of the control. Original miRNAs sequence data are accessible in the NCBI GEO database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE56385). 2.5. Reverse transcription (RT)-PCR and qPCR For the gene expression analysis, total RNAs were extracted using the Trizol reagent (as mentioned in Section 2.4) and reverse transcribed with a PrimeScript® RT reagent kit (TaKaRa, Dalian, China). For the miRNA expression analysis, total RNAs were extracted using a mirVana™ miRNA isolation kit (Ambion, Austin, TX, USA) and reverse transcribed with a miScript II RT kit (Qiagen, Valencia, CA, USA). Target genes or miRNA expressions were detected using a SYBR® Premix Ex TaqTM II (Perfect Real Time) kit (TaKaRa, Dalian, China) on an ABI StepOnePlus PCR system (Applied Biosystems, Foster City, CA, USA). The qPCR condition was 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The expression changes in the genes or miRNAs were calculated as: FC = 2−ΔΔCT. Glyceraldehyde 3-phosphate dehydrogenase (Gapdh) and small U6 were used as the internal control genes to normalize the expression of different genes and miRNAs. All primer sequences used for qPCR examination are shown in the supplementary table (Table S1).

2.3. Cell treatment 2.6. Western blot analysis Normal J1 mESCs were cultured in LIF-containing medium. In treatments without LIF, the normally cultured cells were washed with PBS (140 mM NaCl, 2.7 mM KCI, 56 mM Na2HPO4, and 1.5 mM KH2PO4, pH 7.4), digested with 0.25% trypsin (dissolved in PBS), and then cultured in LIF-free Dulbecco's modified Eagle's medium as described in Section 2.2. SB and retinoic acid (RA) powder were dissolved separately in DMSO and stored at a concentration of 10 mM and 2 mM, respectively. The stored reagents were added directly to the pre-wormed cell culture medium for the J1 mESC treatment. The working concentrations of the reagents were 3 μM SB (unless otherwise indicated) and 1 μM RA. The DMSO-treated cells were used as control to exclude the dissolution influence. 2.4. RNA extraction, whole-genome gene, and miRNA expression examination Total RNAs were extracted from the cells cultured on 6-well plates using a Trizol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. For the expression analysis of the whole-genome gene, the isolated total RNAs were checked and analyzed using an Agilent SurePrint G3 mouse GE 8*60K Microarray (Agilent Technologies) by Shanghai Biotechnology Corporation (Shanghai, China). After removing the probes without apparent signal (called

The differentiation-treated J1 mESCs were washed with PBS and lysed in RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% Na Deoxycholate, and 0.5% Triton X-100) supplemented with complete protease and phosphatase inhibitor (Roche, Mannheim, Germany). The cell lysates were then quantified using a BCA protein analysis kit (Beyotime, Jiangsu, China) and boiled in SDS loading buffers (20% glycerin, 3% SDS, 0.5 M Tris–HCl, and 0.004% bromophenol blue) at 100 °C for 10 min. The boiled cell lysates were fractionated using 10% acrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, MA, USA) at 0.25 A for 2.5 h. After transferring, the membranes were blocked in TBST (pH 7.0, 10 mM Tris–HCl, 150 mM NaCl, and 0.05% Tween-20) containing 10% non-fat milk powder for 2.5 h. The membrane was incubated with the primary antibody at 4 °C overnight. After washing thrice with TBST for 15 min each, the membranes were incubated with the indicated second antibody at room temperature for 2 h. The membrane was washed thrice with TBST for 15 min each and autoradiographed using the SuperSignal West Pico substrate (Pierce/Thermo Scientific, Rockford, IL, USA). 2.7. Alkaline phosphatase (AP) staining The AP activity of J1 cells was detected using a BCIP/NBT alkaline phosphatase color development kit (Beyotime Institute of Biotechnology).

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Briefly, J1 mESCs were washed with PBS and fixed at room temperature for 20 min. After washing thrice with PBS, the fixed cells were incubated in the BCIP/NBT working solution for about 10 min in the dark. 2.8. Pathway confirmation 2.8.1. Luciferase reporter assays Pathway reporter vectors pAP1-TA-luc and pNFκB-TA-luc were purchased from Beyotime Institue of Biotechnology; negative control vectors (pTA-luc, contain a TATA box from the herpes simplex virus thymidine kinase promoter), pNFAT-TA-luc and pISRE-TA-luc were purchased from Clontech Laboratories, Inc. (Mountain View, CA, USA); other vectors including pCRE-TA-luc, pHSE-TA-luc and pGRE-TA-luc were constructed in our laboratory [17]. When examining the indicated signaling pathways activities, special reporter vectors (containing specific pathway transcription factor binding cis-acting DNA sequence—enhancer element) and negative control plasmid pTA-luc (used to determine the background signals) were cotransfected respectively with Renilla luciferase plasmid pRL-SV40 (Promega, Madison, WI, USA, used as an internal control to distinguish differences in transfection efficiency) to the J1 mESCs using LipofectamineTM 2000 Reagent (Invitrogen). After 12 h, transfected J1 mESCs were treated with DMSO (used as control to exclude the influence of the solvent) and SB for additional 24 h. Then, differently treated J1 cells were lysed and luciferase activity was examined using a Dual-Luciferase Repoter Assay System (Promega, Madison, WI, USA) on a VICTOR X5 multilabel plate reader (PerkinElmer, Cetus, Norwalk, USA). 2.8.2. Plasmids construction Tag confused expression plasmids—pCMV–Myc–Smad2, pCMV– HA–Smad3, and pcDNA3.1(+)–Flag–Smad7 were constructed by amplifying and inserting the coding sequence of Smad2, Smad3 and Flag–Smad7 to the enzyme digested pCMV–Myc, pCMV–HA and pcDNA3.1(+) vectors. Primers used for the genes amplification were as follows: Smad2 (sense) 5′-GTGAATTCGGATGTCGTCCATCTTGCCATT CACTCCGC-3′ and (anti-sense) 5′-GACCTCGAGTTACGACATGCTTGAG CATCGCACTG-3′; Smad3 (sense) 5′-CGTGAATTCATATGTCGTCCATCCT GCCCTTCAC-3′ and (anti-sense) 5′-CATGGTACCTTAAGACACACTGGAA CAGCGGATGCTCGG-3′; Smad7 (sense) 5′-CATGGATCCATGGACTACA AGGACGACGATGACAAGTTCAGGACCAAACGATCTG-3′ and (anti-sense) 5′-CATCTCGAGTTACCGGCTGTTGAAGATGACCTC-3′ (underline sequences of the primers are restriction sites, bold sequence within the sense Smad7 primer is Flag tag). 3. Results 3.1. SB maintained the undifferentiated state of ESCs in the presence of LIF mESCs can be successfully maintained in the presence of specific pathway inhibitors [18,19]. The J1 mESCs maintained on gelatincoated dishes with or without LIF (1000 U/mL) were treated with SB to determine the exact contribution of TGF-β signaling inhibition to the undifferentiated states of mESCs. In the presence of LIF, SB significantly compressed the J1 colonies. The J1 mESC colonies with an irregular edge and flatly stretched on feeder-free plates became smooth and tightly protuberant when SB was added. J1 mESCs differentiated spontaneously in the absence of LIF, with the cells around the colonies growing in a neurite-like phenotype. The SB addition did not completely block this differentiation but decreased the number and the length of the neurite-like structures (Fig. 1A). Further examination showed that highly expressed genes in the differentiated embryonic germ layers, including Brachyury/T (mesendoderm), Nestin (ectodermal) and Gata4 (endodermal), were down-regulated by SB. However, the primary

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genes for pluripotency regulation, such as Oct4, Sox2 and Klf4, were not significantly affected by SB (Fig. 1B). These results indicate that SB is a molecular positive for the maintenance of the undifferentiated state of ESCs. Moreover, SB could maintain the undifferentiated state of ESCs through differentiation-inhibiting activities. SB had no significant effect on the key pluripotent-related genes (i.e., Oct4, Sox2, and Klf4) transcription, but greatly reduced the differentiation-related genes (T, Nestin, and Gata4) transcription. 3.2. Transcripts involved in self-renewal and differentiation were regulated by SB The morphological changes in J1 mESCs presented in the culture medium with LIF, LIF-free and SB indicate that SB is a factor positive for the maintenance of undifferentiated state of ESCs. However, more evidence is required to confirm this speculation. The global gene expression profile of J1 mESCs treated with DMSO (solvent of SB used in the control experiment) and SB was separately measured in a microarray experiment to establish the contribution of SB in maintenance of the undifferentiated state of ESCs. After filtration and normalization, 2506 genes of the gene expression profiles (p ≤ 0.05) perturbed by SB were selected. Among the perturbed genes, 1440 were significantly down-regulated (FC ≤ 0.84), and 1066 were up-regulated ((FC ≥ 1.2; Table S2, Supplementary information). Through expression profiling, we found that genes involved in selfrenewal and differentiation of ESCs were affected by SB (Fig. 2A). On the ground of up-regulated genes, Dppa3, Ncoa3, Bmi1, Lin28, Id1 and Id3 were included. Dppa3, which is also known as Stella or Pgc7 and expressed in undifferentiated embryonic cells, embryonic germ cells, and adult germ cell tumors, is a pluripotency marker of ESCs [20]. Ncoa3 is a nuclear receptor coactivator that promotes ESCs selfrenewal corporately in collaboration with Essrrb [21–23]. Bmil, identified as a proto-oncogene, is important for the self-renewal of stem cells, when combined with Oct4, Bmi1 could replace Sox2, Klf4, and c-Myc in inducing the iPS cells from mouse fibroblasts [24]. Lin28 is an RNA-binding protein that directly benefits the mESCs pluripotency through proliferation enhancement [25]. Id1 and Id3 inhibit differentiation members and can enhance the self-renewal of mESCs when collaborated with STAT3 [26]. On the ground of down-regulated genes, T, Lef1, Nes (also named Nestin), Gata4, Pitx2, Zic1, and Sall2 were included. T, Lef1, Nesin, and Gata4 are markers of the three different embryonic layers. Pitx2 is important in endodermal and mesodermal germ layer formation [27]. Zic1 and Sall2 are zinc-finger transcription factors that have an important function in embryonic neuron lineage development [28,29]. RT-qPCR was performed for the part of the indicated genes (Fig. 2B) to confirm the objective reliability of the gene expression changes. A consistent result was obtained. As shown in Fig. 1B, Oct4, Sox2 and Klf4 had no significant expression changes based on expression profiling. Myc (c-Myc) and Nanog transcripts, which are key pluripotency-related transcription factors, were down-regulated (Table S2). The qPCR examination for Myc and Nanog gene transcripts showed a consistent result (Fig. 2C). Nanog has been reported to be a “gatekeeper” of ESC pluripotency, and a low Nanog expression level results in differentiation-prone ESCs [30]. However, previous reports show that Nanog is heterogeneously expressed in ESCs [31,32]. Therefore, a fluctuation of the Nanog gene transcripts in ESCs is acceptable. Western blot for the J1 mESCs cultured in LIF-containing condition showed that SB did not down-regulate the Nanog protein levels after 24 h or longer time (3 days after SB treatment was examined) of treatment (Fig. 2D). Myc is an important regulator of ESCs that directly regulate many embryonic-enriched gene expression [33]. Unlike the Nanog expression patterns, Myc was down-regulated in SB-treated J1 mESCs 3 days after the treatment (Fig. 2D). SB could not block the J1 mESC differentiation under the without LIF condition. The decreased Myc expression in long-time SB-treated J1 mESCs may partly explain this phenomenon. Previous reports show that LIF could directly

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Fig. 1. SB maintains the undifferentiated state of the embryonic stem cells. A. Morphology of J1 mESCs. J1 mESCs cultured in the medium with or without LIF were treated with SB (3 μM) or an equal volume of DMSO for 24 h. B. Relative expression of the typical differentiation- and pluripotent-related genes. Normally cultured J1 mESCs (cell culture medium containing LIF) were treated with SB (3 μM) or an equal volume of DMSO. After 24 h treatment, the cells were lysed with Trizol, RNA was extracted, and qPCR was performed. The qPCR data were normalized to Gapdh expression. Data are presented as mean ± SD of three independent experiments. *p ≤ 0.05; **p ≤ 0.01.

regulate the Myc expression [34], and high Nanog expression could replace LIF to maintain the pluripotent state of ESCs [35]. Moreover, LIF could activate the Nanog expression and the Stat3 binding site at 5.2 kb of the Nanog 5′ promoter region was founded [36]. Along with the morphological changes of J1 mESCs (Fig. 1A), the results showed that SB could not maintain the Nanog expression of mESCs in the absence of LIF and that the Myc expression was down-regulated by SB after long treatment time. These findings indicate that LIF is indispensable in SB to maintain the undifferentiated state of ESCs. Based on the results, SB partially maintains the ESC self-renewal by activating of self-renewal-related genes and inhibiting the regulation of differentiation-related genes. However, whether SB functions mainly on the regulation, self-renewal enhancement, or differentiation inhibition remains unclear, as SB did not significantly increase the main pluripotency related gene expression. Therefore, further analysis is required to resolve this problem. 3.3. SB mainly maintained ESCs by inhibiting their differentiation SB is a specific inhibitor of ALK4, ALK5, and ALK7, which are TGF-β superfamily I ALK receptors, and it specifically blocks the activin, nodal, and TGF-β-mediated Smad2/3 phosphorylation. When SB was added to the cell culture medium, a significant decrease in phosphoSmad2/3 (Fig. 3A) and its direct target genes, including Lefty1/2, Pitx2, and Duxbl [37] was observed (Table 1). However, an activation of Smad1/5/8-related signaling pathway was also observed: Smad7 decreased but Bmp4, Id1, and Id3 were up-regulated (Table 2) [16,38]. Smad7 is an inhibitory Smad proteins, and its overexpression in mESCs significantly inhibits the Smad1/5/8 phosphorylation [39]. However, Bmp4 is an activator of Smad1/5/8, which promotes the ESC pluripotency maintenance by inducing the Id gene expression [26]. Id proteins are negative regulators of cell differentiation that sustains the ESC self-renewal by suppressing differentiation [40–42]. qPCR and Western blot analysis were performed to confirm the reliability of the

results. The results showed that 24 h after SB treatment, the phosphoSmad2/3 target genes decreased significantly, and Lefty1 and Lefty2 were reduced to a nearly undetectable level. The Smad1/5/8 pathwayrelated genes, including Bmp4, Id1, and Id3, significantly increased (Fig. 3B). Corresponding with the up-regulated Id gene transcripts, increased phospho-Smad1/5 levels in the SB-treated J1 mESCs were also examined (Fig. 3C). Thus, SB effectively inhibited the Smad2/3 activation and activated the Id differentiation-suppressing pathway of ESCs, including Bmp, Smads, and Ids. Nine of the Fgf family members found in the expression profiling (Table 3) were all down-regulated by SB. Fgf5 is an ectoderm marker important in mESCs development [43,44]; Fgf10 can induce the differentiation of cardiomyocyte from mouse embryonic or induced pluripotent cells [45]. Fgf8 and Fgf13 are important in neural cell differentiation [46,47]. Fgf4 knocked out ESCs is normally undifferentiated in the presence of LIF, but it lacks ability to differentiate into neural and mesodermal lineage cells [48]. The decreased Fgf4/Erk signaling with downregulated Fgf5 and T expression raises the dynamic ESCs to a highly undifferentiated state [49]. Endogenous FGF-signaling pathway is activated during the early ESCs differentiation [50], but the downregulated Fgf genes suggested that the process of early embryonic cell differentiation was inhibited by SB. The qPCR analysis was performed for the portion of the Fgf genes presented in the expression profiling, and a similar result was obtained (Fig. 3D). Previously, we showed that SB suppressed the neuronal-like structure formation of J1 mESCs cultured under LIF-free conditions. Through the expression profiling, we found that many genes involved in neuronal differentiation were down-regulated by SB. The genes specifically expressed in neuron cells, including Sox1, Ncam1 (also named CD56), Nestin, Bmp6, and neuro-filament light chain (Nefl or NF-L), were down-expressed in SB-treated J1 mESCs unlike in the DMSO mocktreated cells. Moreover, Ests1, Cdx1, Hoxb1, Hoxb4, HSD17b1, growth associated protein 43 (Gap43), RA receptor alpha (Rarа), Egr1, and Tgm2 (Table 4), which are directly regulated by the all-trans RA [51–53],

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Fig. 2. The genes involved in self-renewal and differentiation were regulated by SB. A. Heat map diagram of the representative up-regulated pluripotent genes and down-regulated differentiation genes in J1 mESCs treated with DMSO (control) and SB (3 μM). High expression levels are shown in red and low levels in green. B. the qPCR analysis of the five selected genes was used to confirm the reliability of the microarray data. After 24 h of SB (3 μM) or DMSO treatment, J1 mESCs cultured in the LIF-containing medium were used in the qPCR analysis. The relative expression levels, Gapdh normalized qPCR data, were used to show the expression change of the indicated genes. Data are presented as mean ± SD of three independent experiments. *p ≤ 0.05; **p ≤ 0.01. C. qPCR analysis of Myc (c-Myc) and Nanog presented in SB (3 μM) and DMSO-treated J1 mESCs. Gapdh was used to normalize the template levels. Data presented as the mean ± SD of three independent experiments. *p ≤ 0.05; **p ≤ 0.01. D. Western blot analysis of Nanog and Myc expressed in J1 mESCs. J1 mESCs cultured in the condition with or without LIF were treated with DMSO and SB (3 μM). One or 3 days after treatment, J1 mESCs were lysed with RIPA, quantified using BCA, and subjected to Western blot to analyze the Nanog and Myc expression. Gapdh was used as the loading control.

were down-regulated by SB. This result indicates that SB has the ability to inhibit the neuronal lineage differentiation of J1 mESCs. The qPCR analysis for part of the observed neuronal-related genes confirmed this conclusion (Fig. 3E). RA has been an efficient molecule for neuron-like cell induction [54, 55]. The decreased RA target gene expression by SB treatment encouraged us to determine whether SB inhibits the ESCs differentiation through an RA-related signaling pathway. J1 mESCs initiated their differentiation processes when treated with RA. The J1 colonies began to stretch and obtain a flatter phenotype, and the AP activity of the cells was reduced unlike that of the DMSO mock-treated cells (Fig. 3F). The pretreatment of J1 mESCs with SB did not completely block the RAinduced differentiation but considerably slowed down the process. The colonies in the medium containing SB and RA had a relative compact phenotype and a higher AP activity compared with those in the cells treated with RA only (Fig. 3F). The qPCR examination for Hoxb1 and Hoxb5, which are genes directly regulated by RA, showed that RAinduced Hoxb1 and Hoxb5 expression were down-regulated when the cells were pre-treated with SB (Fig. 3G). These results suggest that SB could suppress the neuronal lineage differentiation of J1 mESCs by inhibiting an RA signaling pathway. The microarray data showed no significant enhancement of the selfrenewal-related pathways or key pluripotency-related gene expression.

These findings indicate that SB contributes mainly to the undifferentiated state of ESCs by inhibiting its differentiation. 3.4. Global down-regulation of J1 mESC miRNA expression was mediated by SB miRNA is a type of single-strand non-coding small RNA (18-25 nucleotides in length) that regulates gene expression through translation inhibition and mRNA destabilization. In ESCs, miRNAs have an important function in gene regulation and early embryonic development [56,57]. The mutual regulation of miRNAs with ESCs transcription factors involves them in the transcriptional regulatory circuitry of ESCs [57–59]. SB primarily maintained the pluripotency state of J1 mESCs by inhibiting differentiation. Thus, the miRNA transcript changes in J1 mESCs cultured in DMSO- and SB-containing medium were further analyzed to confirm this concept. After a 24 h treatment, the small RNAs from J1 mESCs treated independently with DMSO and SB were used for deep-sequence analysis. The total number of reads obtained from three independent replications was 18870345 for DMSO mock-treated J1 cells and 21947952 for SB-treated J1 cells. After aligning with a miRBase and further screening (p ≤ 0.01, FC ≥ 2 or ≤0.5), a total of 114 mature miRNAs modified by SB were selected (Table S2). The miRNA data mainly showed down-

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Fig. 3. A. Effects of SB on the Smad2/3 activity. J1 mESCs cultured in LIF-containing medium were treated with DMSO, 3 μM SB, and 5 μM SB for 24 h. After 24 h of DMSO and SB treatment, J1 mESCs were lysed with RIPA, quantified using BCA and subjected to Western blot to analyze the phospho-Smad2/3 and Smad2 levels. Smad2 was used as the loading control. B. qPCR analysis for the representative phospho-Smad2/3 regulated gene expression. J1 mESCs cultured in LIF-containing medium were used for qPCR analysis 24 h after SB (3 μM) or DMSO addition. Relative expression levels, Gapdh normalized qPCR data were used to show the expression change of the indicated genes. Data are presented as mean ± SD of three independent experiments. *p ≤ 0.05; **p ≤ 0.01. C. Western blot analysis of the phospho-Smad1/5 levels in J1 mESCs treated with DMSO, 3 μM and 5 μM SB. J1 mESCs were cultured in the medium with LIF, DMSO, or SB for 24 h. Smad1 was used as the loading control. D and E. Effects of SB on the Fgf genes (D) and the representative RA-regulated genes (E) expression. J1 mESCs maintained in normal culture medium were treated with 3 μM SB and equal volume of DMSO. After 24 h of treatment, the cells were processed for qPCR analysis. F. SB partially inhibited the RAinduced J1 mESC differentiation. J1 mESCs were maintained in normal culture medium with DMSO or SB (3 μM). One day after incubation with DMSO and SB, RA (1 μM) was added in the J1 mESC culture. After 24 h from the RA addition, differently treated J1 mESCs were processed for AP staining and photographed. G. SB compromised RA induction of Hoxb1 and Hoxb5. One day after DMSO and SB (3 μM) treatment, J1 mESCs were culture further in the medium containing DMSO, SB, DMSO + RA (RA, 1 μM), and SB + RA for another 24 h. Transcripts of Hoxb1 and Hoxb5 in the treated J1 mESCs were analyzed using qPCR. Gapdh was used to normalize the template levels. Data are presented as mean ± SD of three independent experiments. *p ≤ 0.05; **p ≤ 0.01.

regulation with only four up-regulated miRNAs (i.e., mmu-miR-122-5p, mmu-miR-3096b-5p, mmu-miR-3096-5p and mmu-miR-3471). The 110 down-regulated miRNAs that responded directly to phosphoSmad2/3 were mmu-miR-181d-5p, mmu-miR-7a-5p, mmu-miR-181c5p, mmu-miR-323-3p, mmu-miR-744-5p, mmu-miR-382-5p, mmumiR-672-5p, mmu-miR-185-5p, mmu-miR-341-3p and mmu-miR382-3p (Table 5) [60]. The down-regulated phospho-Smad2/3 response of miRNAs confirmed that SB inhibited the Smad2/3 activities. Moreover, SB influenced the global genome transcription at the mRNA and miRNA transcription levels. We noted that miRNAs, which were positive for pluripotency gene inhibition and ESCs differentiation, were down-regulated by SB aside from the phosho-Smad2/3 targets. miRNAs included in the pluripotent inhibition were let-7 family members, mir-145, mir-134, mir-125, and mir-181. The let-7 family members that antagonize the ESCs cycleregulation miRNAs could suppress the self-renewal of ESCs [61]. However, mir-145, mir-134, mir-125, and mir-181, down-regulate the

pluripotency of ESCs by inhibiting the pluripotent-related gene expression [62–64]. The differentiation-promoting miRNAs included mir-124, mir-9, and some RA response miRNAs, such as mir-23, mir10a, mir-134, mir-296, and mir-470. The important regulators for neuron fate determination are mir-124, which induces neurogenesis by suppressing the small C-terminal domain phosphatase 1, and mir9, which promotes neuron cell differentiation of mouse bone mesenchymal stem cells by notch signaling [65–67]. Similarly, mir-23, mir-10a, mir-296, and mir-470 are also associated with neuronal cells formation, and their expression can be enhanced by RA during the ESCs differentiation induction [59]. Remarkably, mir-10a and mir196a, which are located collinearly with Hoxb clusters, were also down-regulated in SB-treated J1 mESCs. These results and the finding in which many RA response genes were down-regulated by SB suggest that SB could resist endogenous RA response transcript expression; this suggestion could be a partial reason for the inhibition of SB in stem cell differentiation. The qPCR analysis for the portion of the indicated

J. Du et al. / Cellular Signalling 26 (2014) 2107–2116 Table 1 Fold change of phosho-Smad2/3 directing targets in SB treated J1 mESCs compared with that of DMSO treated control cells. Gene name

Fold change

p-Value

Duxb1 Cnpy1 Camk2n1 GalNAcs-6ST Ubr7 Abcg2 AW548124 B3galnt1 Bcar3 Bhlhb8 Ccnd2 Epha2 Fgf15 Lefty1 Lefty2 Mcl1 Nodal Notch3 Nphs1 Nxn Pitx2 Plekha2 Rasd2 Rhob Ski Smad7 SnoN Tmem63a Tmepai Zfp423 Ctgf

0.0963 0.2916 0.5446 0.1699 0.5191 0.3673 0.1695 0.5022 0.4754 0.4748 0.3509 0.2327 0.0084 0.0017 0.0379 0.6624 0.6448 0.4673 0.4605 0.5686 0.003 0.6408 0.4369 0.6132 0.66 0.0448 0.3997 0.0951 0.171, 0.1687 0.6662, 0.5693 0.5285

0.0012 0.0020 1.00E−04 0.0024 4.00E−04 0.0011 0 0.0019 3.00E−04 0 8.00E−04 4.00E−04 0.0887 4.00E−04 3.00E−04 0.0011 0.0016 5.00E−04 0.005 9.00E−04 1.00E−04 0.0019 0 0.0024 0.0266 0 3.0E−4, 0.0 0 1.0E−4, 0.0022 0.0010, 0.0015 4.00E−04

miRNAs was performed to confirm the accuracy of the deep-sequence data (Fig. 4). A consistent result was obtained. Therefore, a global decreased in miRNAs indicates that SB functions mainly by inhibiting differentiation. SB inhibited the phospho-Smad2/ 3 regulated transcripts, differentiation-related factors, and ESCs selfrenewal inhibitors.

3.5. Ways of SB in maintaining mESCs undifferentiated state Based on the above results, presumable ways of SB in maintaining the undifferentiated state of mESCs are shown in Fig. 5A. To verify what Fig. 5A has shown, pathway reporter vectors were used to examine the response of the indicated signaling ways to SB treatment. As Fig. 5B shows, treatment of vector-transfected cells with 3 μM SB significantly decreased TGF-β mediated reporter gene expression (pAP1-luc, AP1) [68]. Erk and RA response reporter activities (pSRE-Luc, SRE and pISRE-Luc, ISRE) [69,70] were also down-regulated, other pathway reporter gene expression, however, did not significantly influenced by SB. These results confirmed that SB did inhibit TGF-β, Erk1/Erk2 and RA signaling pathways.

Table 2 Activated Smad1/5/8 pathway regulating and regulated genes. Gene name

Fold change

p-Value

Bmp4 Id1 Id3 Id4 Msx2 Eif4a2 Smad6 Smad7

1.9168 1.9101 1.541 0.7246 1.275 0.8032 0.6565 0.0448

0.0097 7.00E−04 0 0.0214 0.0216 0.0189 0 0

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Table 3 Expression changes of fibroblast growth factors founded in SB treated J1 mESCs. Gene name

Fold change

p-Value

Fgf5 Fgf10 Fgf8 Fgf13 (Fhf2) Fgf4 Fgf17 Fgf18 Fgf15 (Fgf19) Fgf21

0.1117 0.4131 0.2162, 0.0147 0.6052 0.7824 0.5024 0.787 0.0887 0.2569

0.0086 0.001 1.0E−4, 0.3421 0.0374 6.00E−04 0.0042 0.0327 0.0084 0.014

Furthermore, SB down-regulated Smad7 expression was increased in Smad2 and Smad3 overexpressed cells when compared with that of control vector (pCMV–Myc and pCMV–HA) transfected J1 mESCs (Fig. 5C and D). Smad7 overexpression reduced not only SB induced Smad1/5/8 phosphorylation but also Id gene expression that upregulated in SB containing medium (Fig. 5E and F). These results together indicated that SB activated phospho-Smad1/5/8–Ids pathway through down-regulation of Smad7 expression. As we have shown previously, SB slowed down the RA initiated J1 mESCs differentiation process, and RA induced the increase of differentiation-related genes (i.e., Hoxb1 and Hoxb5) which were down-regulated by SB, these combined with what we have shown above suggested that the ways SB functions on mESCs as shown in Fig. 5A existed.

4. Discussion SB specifically inhibits the function of TGF-β superfamily Type I receptor (i.e., ALK4, ALK5, and ALK7) and thus specifically inhibits the Smad2/3 activities as well. In this study, microarray and deepsequence data were used cooperatively to explore the way of SB in maintaining the undifferentiated state of mESCs. The data indicated that SB inhibited the Smad2/3 activity. The Smad2/3 targets, such as genes and miRNAs, were significantly down-regulated in the J1 mESCs cultured in SB-added medium. Moreover, SB regulated the gene transcripts related to self-renewal and differentiation but mainly functioned by inhibiting the differentiation-related transcripts. The analysis of SBregulated genes or miRNAs through Western blot and qPCR showed

Table 4 Down-regulated RA-response genes in SB treated J1 mESCs compared with that of DMSO mock treated J1 mESCs. Gene name

Fold change

p-Value

Rara Gap43 Lgals3 Acta2 App Cdkn1a Myc Ptgs1 Hoxb4 Serpinh1 Egr1 Lgals3 Lmna Ngfr Cdkn1a Ngfr Muc3 Efnb1 Tgm2 Hsd17b1

0.6884, 0.5524 0.5754 0.7216 0.6419 0.76 0.6874 0.488 0.2873 0.3101 0.5549 0.3225 0.7216 0.8129 0.4317 0.6874 0.4317 0.7949 0.2333 0.6826, 0.8082 0.4707

0.0164, 0.0023 3.00E−04 0.0011 4.00E−04 0.0022 0.041 2.00E−04 0.0055 0.0115 2.00E−04 9.00E−04 0.0011 0.0052 2.00E−04 0.041 2.00E−04 5.00E−04 0 0.0049, 0.0217 0.0393

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Table 5 Expression changes of phospho-Smad2/3-response miRNAs significantly regulated by SB compared with that of DMSO treated J1 mESCs. miRNA name

Fold change

p-Value

mmu-miR-181d-5p mmu-miR-7a-5p mmu-miR-181c-5p mmu-miR-185-5p mmu-miR-323-3p mmu-miR-744-5p mmu-miR-382-5p mmu-miR-672-5p mmu-miR-341-3p mmu-miR-382-3p

0.314551 0.436915 0.371769 0.469911 0.45929 0.468006 0.463813 0.447 0.17559 0.451541

0 1.96E−149 1.38E−186 0 0 0 0 0 0 8.55E−303

that our results were accurate. The consistent change in mRNA transcripts and miRNAs, such as Lin28 and let-7, further confirmed the consistency of our data. Microarray data showed that Lin28 was up-regulated, whereas let-7 miRNA family members in the deepsequence data were down-regulated in the SB-cultured J1 mESCs than in the DMSO-treated J1 cells. In mESCs, Lin28 and let-7 family members mutually regulate each other's expression. Lin28 inhibits the let-7 family member maturation and subsequently inhibits the let-7 family member-mediated mESCs differentiation [71]. However, the let-7 family members can directly regulate the Lin28 gene expression [72]. The combination of Lin28 expression with let-7 family members in SBtreated J1 mESCs confirmed that they regulated each other's expression and showed that the microarray data and deep-sequence data were acceptable. The Smad2/3-mediated signaling is indispensable for hESCs pluripotency maintenance [73]. SB-mediated inhibition of the Smad2/ 3 signal transduction enhances the hESCs differentiation [13]. Smad2/ 3 balances the hESCs self-renewal and differentiation through a PI3K/ Akt-dependent activity, in which Smad2/3 promotes the self-renewal of cells when PI3K/Akt is activated and Smad2/3 promotes pluripotent cells differentiation when a low PI3K/Akt activity is presented [74]. The Smad2/3-signaling pathway is dispensable and detrimental to mESCs pluripotency maintenance, which is different from hESCs. The inhibition of the Smad2/3 activity by SB has been proved to replace Sox2 and/or c-Myc to induce the formation of pluripotent cells with other inducing factors. SB functions mainly on the last induction process when it is used to induce the pluripotent cells [15,75]. Moreover, SBinduced TGF-β signaling inhibition protects the differentiation triggers of pluripotent cells and thus promotes the ground state of the cells [14]. These studies indicate that Smad2/3-mediated TGF-β signaling

pathway can protect the pluripotent-to-differentiation method of mESCs. The activated Smad2/3 promotes the pluripotency cells toward differentiation, whereas the inhibited Smad2/3 function promotes the differentiated cells toward obtaining and maintaining pluripotency. In this study, a consistent conclusion was obtained. SB mediated the Smad2/3 signal inhibition to maintain the J1 mESC pluripotent state and blocked the differentiation phenotype of J1 mESCs. The J1 colonies cultured in traditional LIF-containing medium became compactly connected and exhibited a definite periphery when Smad2/3 signal was inhibited. The results showed that SB maintained the undifferentiated state of J1 mESCs. Thus, microarray experiment and deep-sequence analysis were used to elucidate the mechanism of SB in maintaining pluripotency of the mESC. Data obtained from the microarray experiment and deep-sequence analysis showed that the addition of SB significantly decreased the phospho-Smad2/3 response target expressions. However, the key pluripotent factor expression (i.e., Klf4, Sox2, Oct4, and Nanog) that directly regulates the ESCs pluripotency did not significantly increase. SB could have functioned not mainly on the selfrenewal promotion but on a differentiation regulation, and this speculation was confirmed by the microarray and deep-sequence data. Many differentiation-related genes, particularly neuronal lineage differentiation genes such as Sox1, Nestin, and Hoxb1, were down-regulated by SB. The miRNAs positive for ESCs differentiation were also downregulated by SB. The pluripotent stem cells that were cultured in vitro in the presence of LIF still differentiated spontaneously into neuronal cell lineage. Researchers have shown that Bmp4 could inhibit the “default differentiation” of pluripotent cells toward neuronal precursors through an Id gene induction method [42,76]. The Fgf genes, including Fgf4 and Fgf5 that autocrine by pluripotent cells, could induce the neural precursor formation of the cells by activating the Erk1/2 signal pathway [48,49, 77,78]. In the present study, we found that SB significantly inhibited the Smad2/3 activity, increased the Bmp4-phospho-Smad1/5/8-Ids activity, decreased the Fgf gene expressions, and suppressed Erk1/2 activity, these all contribute to suppressing the neuronal differentiation of mESC. Moreover, SB decreased some intrinsic RA response gene expressions, RA response reporter gene expression was also down-regulated by SB. J1 mESCs pretreated with SB could be partly resistant to RAinduced neuronal lineage. The differentiation morphology of the J1 colonies appeared later in the SB-pretreated J1 mESCs, and the gene responses to RA induction were reduced in SB-added condition. All results indicated that SB suppressed the J1 mESC “default” differentiation by activating the differentiation-inhibiting process and suppressing the differentiation-inducing activity. It is noteworthy that SB reduces RA activity not by interfering the action between Rarа and RA—when Rarа was pre-introduced into J1 mESCs, SB could not reduce RA up-regulated reporter gene expression (data not shown). All together, our results indicated that inhibiting Tgf-β signaling pathway by SB contributed to the maintenance of mESCs pluripotent state. 5. Conclusions On the basis of the morphology and transcript changes in J1 mESCs cultured with or without SB, we found that SB promoted the maintenance of the undifferentiated state of ESCs mainly through the differentiation-inhibiting process. SB inhibited the Fgf-induced “default” differentiation of ESCs; RA-induced differentiation pathways partially comprised by SB; and many differentiation-related genes, particularly neuronal-related genes, were down-regulated by SB.

Fig. 4. Expression changes of miRNAs in SB-treated J1 mESCs compared with those in the control cells. miRNAs expressed in J1 mESCs treated with DMSO or SB for 24 h were isolated and quantified through a qPCR experiment. The qPCR data of different miRNAs were normalized to U6. Data are presented as mean ± SD of three independent experiments. *p ≤ 0.05; **p ≤ 0.01.

Conflicts of Interests The authors have declared that no conflicts of interest exist.

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Fig. 5. A. Schematic diagram of the SB functions in maintaining the undifferentiated state of mESCs. Genes shown in red color were down-regulated by SB, and genes in green were upregulated by SB. → means active, ⊥ means inactive. B. Relative luciferase activity of J1 mESCs transfected with differential report vectors and pRL-SV40. 12 h after transfection, J1 mESCs were treated with 3 μM SB or equal volume DMSO for additional 24 h, and then used to examine the luciferase activity. Origin data obtained were firstly normalized to the Renilla luciferase, and then normalized to the negative control reporter's luciferase activity. C. Examination of tag confused Smad2, Smad3 and Smad7 expression. Thirty-six hours after transfection, J1 mESCs with different vectors, pCMV–Myc (Myc), pCMV–Myc–Smad2 (Myc–Smad2), pCMV–HA (HA), pCMV–HA–Smad3 (HA–Smad3), pcDNA3.1(+) and pcNDA3.1(+)–Flag–Smad7 (Flag–Smad7), were Western blot analyzed using different tag antibodies. D. Smad7 expression in differential treated J1 mESCs. After 12 h of plasmids treatment, J1 mESCs were further incubated in SB (3 μM) or equal volume DMSO containing medium for 24 h. qPCR was performed to examine the Smad7 gene expression. Gapdh was used to normalize the template levels. Data are presented as mean ± SD of three independent experiments. *p ≤ 0.05; **p ≤ 0.01. E. Influence of Smad7 on SB activated Smad1/5/8 phosphorylation. Twelve hours after transfection, J1 mESCs carrying pcDNA3.1(+) and pcDNA3.1(+)–Flag–Smad7 (Flag–Smad7) were further incubated in SB (3 μM) or equal volume of DMSO-containing medium for 24 h. Upper panel: Western blot analysis of phosho-Smad1/5 and Smad1 (Smad1 were used as the loading control). Bottom panel: relative intensity of phosho-Smad1/5 compared with that of Smad1 (the Western blot images were analyzed using the Image J). F. Influence of Smad7 expression on Id1 and Id3 gene expression. J1 mESCs pre-transfected with pcDNA3.1(+) and pcDNA3.1(+)–Flag–Smad7 (Flag–Smad7) for 12 h were SB (3 μM) and equal volume DMSO treated for additional 24 h. Id1 and Id3 expression was qPCR examined, Gapdh normalized, and presented as mean ± SD of three independent experiments. **p ≤ 0.01.

Contributions Zekun Guo and Juan Du conceived and designed the experiments. Yongyan Wu and Zhiying Ai collected samples used for microarray and deep-sequence experiments. Xiaoyan Shi, Linlin Chen and Juan Du performed Western blot and qPCR confirmation with Juan Du who wrote the paper.

References [1] [2] [3] [4] [5] [6] [7]

Acknowledgments

[8] [9]

We thank Yuan Gao PhD for his assistance in drawing the heat map. This work was supported by the National Natural Science Foundation of China (grant number 31172279).

[10] [11] [12]

Appendix A. Supplementary data [13]

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.cellsig.2014.06.002.

[14]

G.R. Martin, Proc. Natl. Acad. Sci. U. S. A. 78 (1981) 7634–7638. H.S. Bernstein, D. Srivastava, Pediatr. Res. 71 (2012) 491–499. M.P. Lutolf, P.M. Gilbert, H.M. Blau, Nature 462 (2009) 433–441. A.V. Molofsky, R. Pardal, S.J. Morrison, Curr. Opin. Cell Biol. 16 (2004) 700–707. H. Niwa, Cell Struct. Funct. 26 (2001) 137–148. M. Amit, M.K. Carpenter, M.S. Inokuma, C.P. Chiu, C.P. Harris, M.A. Waknitz, J. Itskovitz-Eldor, J.A. Thomson, Dev. Biol. 227 (2000) 271–278. A.G. Smith, J.K. Heath, D.D. Donaldson, G.G. Wong, J. Moreau, M. Stahl, D. Rogers, Nature 336 (1988) 688–690. S. Chen, J.T. Do, Q. Zhang, S. Yao, F. Yan, E.C. Peters, H.R. Scholer, P.G. Schultz, S. Ding, Proc. Natl. Acad. Sci. 103 (2006) 17266–17271. N. Sato, L. Meijer, L. Skaltsounis, P. Greengard, A.H. Brivanlou, Nat. Med. 10 (2003) 55–63. M. Buehr, S. Meek, K. Blair, J. Yang, J. Ure, J. Silva, R. McLay, J. Hall, Q.L. Ying, A. Smith, Cell 135 (2008) 1287–1298. G.J. Inman, F.J. Nicolas, J.F. Callahan, J.D. Harling, L.M. Gaster, A.D. Reith, N.J. Laping, C.S. Hill, Mol. Pharmacol. 62 (2002) 65–74. M. Van der Jeught, B. Heindryckx, T. O'Leary, G. Duggal, S. Ghimire, S. Lierman, N. Van Roy, S.M. Chuva de Sousa Lopes, T. Deroo, D. Deforce, P. De Sutter, Hum. Reprod. 29 (2013) 41–48. A. Mahmood, L. Harkness, H.D. Schrøder, B.M. Abdallah, M. Kassem, J. Bone Miner. Res. 25 (2010) 1216–1233. S.-N. Hassani, M. Totonchi, A. Sharifi-Zarchi, S. Mollamohammadi, M. Pakzad, S. Moradi, A. Samadian, N. Masoudi, S. Mirshahvaladi, A. Farrokhi, B. Greber, M.J.

2116

[15] [16] [17] [18] [19] [20] [21] [22] [23] [24]

[25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36]

[37] [38] [39] [40] [41] [42] [43] [44] [45] [46]

J. Du et al. / Cellular Signalling 26 (2014) 2107–2116 Araúzo-Bravo, D. Sabour, M. Sadeghi, G.H. Salekdeh, H. Gourabi, H.R. Schöler, H. Baharvand, Stem Cell Rev. Rep. 10 (2014) 13–16. N. Maherali, K. Hochedlinger, Curr. Biol. 19 (2009) 1718–1723. K.E. Galvin, E.D. Travis, D. Yee, T. Magnuson, J.L. Vivian, J. Biol. Chem. 285 (2010) 19747–19756. X. Shi, Y. Wu, Z. Ai, X. Liu, L. Yang, J. Du, J. Shao, Z. Guo, Y. Zhang, Cell. Physiol. Biochem. 32 (2013) 459–475. N. Sato, L. Meijer, L. Skaltsounis, P. Greengard, A.H. Brivanlou, Nat. Med. 10 (2004) 55–63. W. Xiong, Y. Gao, X. Cheng, C. Martin, D. Wu, S. Yao, M.-J. Kim, Y. Liu, J. Vis. Exp. 13 (2009) 1550. J. Bowles, R.P. Teasdale, K. James, P. Koopman, Cytogenet. Genome Res. 101 (2003) 261–265. M. Percharde, V. Azuara, Cell Cycle 12 (2013) 195–196. Z. Wu, M. Yang, H. Liu, H. Guo, Y. Wang, H. Cheng, L. Chen, J. Biol. Chem. 287 (2012) 38295–38304. M. Percharde, F. Lavial, J.H. Ng, V. Kumar, R.A. Tomaz, N. Martin, J.C. Yeo, J. Gil, S. Prabhakar, H.H. Ng, M.G. Parker, V. Azuara, Genes Dev. 26 (2012) 2286–2298. J.H. Moon, J.S. Heo, J.S. Kim, E.K. Jun, J.H. Lee, A. Kim, J. Kim, K.Y. Whang, Y.K. Kang, S. Yeo, H.J. Lim, D.W. Han, D.W. Kim, S. Oh, B.S. Yoon, H.R. Scholer, S. You, Cell Res. 21 (2011) 1305–1315. B. Xu, K. Zhang, Y. Huang, RNA 15 (2009) 357–361. Q.L. Ying, J. Nichols, I. Chambers, A. Smith, Cell 115 (2003) 281–292. M. Faucourt, E. Houliston, L. Besnardeau, D. Kimelman, T. Lepage, Dev. Biol. 229 (2001) 287–306. R. Pincheira, D.B. Donner, Ann. N. Y. Acad. Sci. 1144 (2008) 53–55. J. Aruga, Mol. Cell. Neurosci. 26 (2004) 205–221. L. Hyslop, M. Stojkovic, L. Armstrong, T. Walter, P. Stojkovic, S. Przyborski, M. Herbert, A. Murdoch, T. Strachan, M. Lako, Stem Cells 23 (2005) 1035–1043. A.M. Singh, T. Hamazaki, K.E. Hankowski, N. Terada, Stem Cells 25 (2007) 2534–2542. M.A. Goodell, T. Kalmar, C. Lim, P. Hayward, S. Muñoz-Descalzo, J. Nichols, J. GarciaOjalvo, A. Martinez Arias, PLoS Biol. 7 (2009) e1000149. B.L. Kidder, J. Yang, S. Palmer, PLoS One 3 (2008) e3932. P. Cartwright, Development 132 (2005) 885–896. I. Chambers, D. Colby, M. Robertson, J. Nichols, S. Lee, S. Tweedie, A. Smith, Cell 113 (2003) 643–655. A. Suzuki, A. Raya, Y. Kawakami, M. Morita, T. Matsui, K. Nakashima, F.H. Gage, C. Rodriguez-Esteban, J.C. Izpisua Belmonte, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 10294–10299. K.L. Lee, S.K. Lim, Y.L. Orlov, Y. Yit, PLoS Genet. 7 (2011) e1002130. A. Hollnagel, V. Oehlmann, J. Heymer, U. Ruther, A. Nordheim, J. Biol. Chem. 274 (1999) 19838–19845. Y. Nishimura, A. Kurisaki, M. Nakanishi, K. Ohnuma, N. Ninomiya, S. Komazaki, S. Ishiura, M. Asashima, Biochem. Biophys. Res. Commun. 401 (2010) 1–6. J.D. Norton, R.W. Deed, G. Craggs, F. Sablitzky, Trends Cell Biol. 8 (1998) 58–65. Y. Yokota, S. Mori, J. Cell. Physiol. 190 (2002) 21–28. Q.L. Ying, Nichols, Chambers I and Smith A, Cell 115 (2003) 281–292. J.M. Hebert, M. Boyle, G.R. Martin, Development 112 (1991) 407–415. O. Haub, M. Goldfarb, Development 112 (1991) 397–406. S.S. Chan, H.J. Li, Y.C. Hsueh, D.S. Lee, J.H. Chen, S.M. Hwang, C.Y. Chen, E. Shih, P.C. Hsieh, PLoS One 5 (2010) e14414. C. Wang, C. Xia, W. Bian, L. Liu, W. Lin, Y.G. Chen, S.L. Ang, N. Jing, Mol. Biol. Cell 17 (2006) 3075–3084.

[47] S. Nishimoto, E. Nishida, J. Biol. Chem. 282 (2007) 24255–24261. [48] T. Kunath, M.K. Saba-El-Leil, M. Almousailleakh, J. Wray, S. Meloche, A. Smith, Development 134 (2007) 2895–2902. [49] F. Lanner, J. Rossant, Development 137 (2010) 3351–3360. [50] M.A. Cohen, P. Itsykson, B.E. Reubinoff, Dev. Biol. 340 (2010) 450–458. [51] S. Mani, J. Schaefer, K.F. Meiri, Brain Res. 853 (2000) 384–395. [52] J.E. Balmer, J. Lipid Res. 43 (2002) 1773–1808. [53] J.C. Zhao, L.X. Zhang, Y. Zhang, Y.F. Shen, J. Cell. Physiol. 227 (2012) 2645–2653. [54] G. Bain, D. Kitchens, M. Yao, J.E. Huettner, D.I. Gottlieb, Dev. Biol. 168 (1995) 342–357. [55] G. Bain, W.J. Ray, M. Yao, D.I. Gottlieb, Biochem. Biophys. Res. Commun. 223 (1996) 691–694. [56] Y. Wang, D.N. Keys, J.K. Au-Young, C. Chen, J. Cell. Physiol. 218 (2009) 251–255. [57] C. Melton, R. Blelloch, Adv. Exp. Med. Biol. 695 (2010) 105–117. [58] A. Marson, S.S. Levine, M.F. Cole, G.M. Frampton, T. Brambrink, S. Johnstone, M.G. Guenther, W.K. Johnston, M. Wernig, J. Newman, J.M. Calabrese, L.M. Dennis, T.L. Volkert, S. Gupta, J. Love, N. Hannett, P.A. Sharp, D.P. Bartel, R. Jaenisch, R.A. Young, Cell 134 (2008) 521–533. [59] Y. Tay, J. Zhang, A.M. Thomson, B. Lim, I. Rigoutsos, Nature 455 (2008) 1124–1128. [60] A.J. Cooney, N. Redshaw, C. Camps, V. Sharma, M. Motallebipour, M. Guzman-Ayala, S. Oikonomopoulos, E. Thymiakou, J. Ragoussis, V. Episkopou, PLoS One 8 (2013) e55186. [61] C. Melton, R.L. Judson, R. Blelloch, Nature 463 (2010) 621–626. [62] N. Xu, T. Papagiannakopoulos, G. Pan, J.A. Thomson, K.S. Kosik, Cell 137 (2009) 647–658. [63] Y.M. Tay, W.L. Tam, Y.S. Ang, P.M. Gaughwin, H. Yang, W. Wang, R. Liu, J. George, H.H. Ng, R.J. Perera, T. Lufkin, I. Rigoutsos, A.M. Thomson, B. Lim, Stem Cells 26 (2008) 17–29. [64] A. O'Loghlen, Ana M. Muñoz-Cabello, A. Gaspar-Maia, H.-A. Wu, A. Banito, N. Kunowska, T. Racek, Helen N. Pemberton, P. Beolchi, F. Lavial, O. Masui, M. Vermeulen, T. Carroll, J. Graumann, E. Heard, N. Dillon, V. Azuara, Ambrosius P. Snijders, G. Peters, E. Bernstein, J. Gil, Cell Stem Cell 10 (2012) 33–46. [65] A.M. Krichevsky, K.C. Sonntag, O. Isacson, K.S. Kosik, Stem Cells 24 (2006) 857–864. [66] L. Jing, Y. Jia, J. Lu, R. Han, J. Li, S. Wang, T. Peng, Y. Jia, Neuroreport 22 (2011) 206–211. [67] J. Visvanathan, S. Lee, B. Lee, J.W. Lee, S.K. Lee, Genes Dev. 21 (2007) 744–749. [68] Y. Zhang, X.H. Feng, R. Derynck, Nature 394 (1998) 909–913. [69] H. Gille, T. Strahl, P.E. Shaw, Curr. Biol. 5 (1995) 1191–1200. [70] S. Matikainen, A. Lehtonen, T. Sareneva, I. Julkunen, Leuk. Lymphoma 30 (1998) 63–71. [71] J.P. Hagan, E. Piskounova, R.I. Gregory, Nat. Struct. Mol. Biol. 16 (2009) 1021–1025. [72] X. Zhong, N. Li, S. Liang, Q. Huang, G. Coukos, L. Zhang, J. Biol. Chem. 285 (2010) 41961–41971. [73] D. James, Development 132 (2005) 1273–1282. [74] A.M. Singh, D. Reynolds, T. Cliff, S. Ohtsuka, A.L. Mattheyses, Y. Sun, L. Menendez, M. Kulik, S. Dalton, Cell Stem Cell 10 (2012) 312–326. [75] J.K. Ichida, J. Blanchard, K. Lam, E.Y. Son, J.E. Chung, D. Egli, K.M. Loh, A.C. Carter, F.P. Di Giorgio, K. Koszka, D. Huangfu, H. Akutsu, D.R. Liu, L.L. Rubin, K. Eggan, Cell Stem Cell 5 (2009) 491–503. [76] V. Tropepe, S. Hitoshi, C. Sirard, T.W. Mak, J. Rossant, D. van der Kooy, Neuron 30 (2001) 65–78. [77] M.P. Stavridis, J.S. Lunn, B.J. Collins, K.G. Storey, Development 134 (2007) 2889–2894. [78] Q.-L. Ying, M. Stavridis, D. Griffiths, M. Li, A. Smith, Nat. Biotechnol. 21 (2003) 183–186.

Mechanism of SB431542 in inhibiting mouse embryonic stem cell differentiation.

SB431542 (SB) is an established small molecular inhibitor that specifically binds to the ATP binding domains of the activin receptor-like kinase recep...
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