GATA4 caused atrial and ventricular septal defects and pulmonary valve stenosis in humans. GATA4 encodes a cardiac transcription factor, and when deleted in mice it results in cardiac bifida and lethality by embryonic day (E)9.5. In vitro, the mutant GATA4 protein has a reduced DNA binding affinity and transcriptional activity and abolishes a physical interaction with TBX5, a transcription factor critical for normal heart formation. To characterize the mutation in vivo, we generated mice harboring the same mutation, Gata4 G295S. Mice homozygous for the Gata4 G295S mutant allele have normal ventral body patterning and heart looping, but have a thin ventricular myocardium, single ventricular chamber, and lethality by E11.5. While heterozygous Gata4 G295S mutant mice are viable, a subset of these mice have semilunar valve stenosis and small defects of the atrial septum. Gene expression studies of homozygous mutant mice suggest the G295S protein can sufficiently activate downstream targets of Gata4 in the endoderm but not in the developing heart. Cardiomyocyte proliferation deficits and decreased cardiac expression of CCND2, a member of the cyclin family and a direct target of Gata4, were found in embryos both homozygous and heterozygous for the Gata4 G295S allele. To further define functions of the Gata4 G295S mutation in vivo, compound mutant mice were generated in which specific cell lineages harbored both the Gata4 G295S mutant and Gata4 null alleles. Examination of these mice demonstrated that the Gata4 G295S protein has functional deficits in early myocardial development. In summary, the Gata4 G295S mutation functions as a hypomorph in vivo and leads to defects in cardiomyocyte proliferation during embryogenesis, which may contribute to the development of congenital heart defects in humans.Defects of atrial and ventricular septation are the most frequent form of congenital heart disease, accounting for almost 50% of all cases. We previously reported that a heterozygous G296S missense mutation of  GATA4, which was associated with atrial and ventricular septal defects in a large kindred. The disease-causing mutation has a spectrum of biochemical deficits affecting both DNA binding and protein–protein interactions. Here, we report the generation and phenotypic characterization of mice harboring the orthologous mutation in Gata4 (G295S). While homozygous mutant mice display embryonic lethality and cardiac defects, the phenotype is less severe than Gata4-null mice. A subset of Gata4 G295S heterozygote mice display a persistent interatrial communication  and stenosis of the semilunar valves. Molecular characterization of the mutant mice suggests that the Gata4 G295S mutant protein results in diminished expression of Gata4 target genes in the heart and functional deficits in cardiomyocyte proliferation. Thus, cardiomyocyte proliferation defects may contribute to defects of cardiac septation found in humans with GATA4 mutations.Cardiac malformations occur due to abnormal heart development and are the most prevalent human birth defect. Defects of atrial and ventricular septation are the most common type of congenital heart defect and are the result of incomplete closure of the atrial and ventricular septa, a process required for formation of a four-chambered heart. The molecular mechanisms that underlie atrial and ventricular septal defects are unknown. We previously published a highly penetrant autosomal dominant mutation (G296S) in  Congenital heart defects (CHD) are the most prevalent of all human birth defects with an estimated incidence of 6–8 per 1,000 live births Tbx5 haploinsufficiency in mice accurately mimics the phenotype found in patients with Holt-Oram syndrome Nkx2-5 in mice causes developmental arrest during heart tube looping, haploinsufficiency of Nkx2-5 results in only subtle defects of atrial septation Gata4 display embryonic lethality and defects in ventral morphogenesis associated with failure to form a single ventral heart tube Monogenic etiologies for atrial and ventricular septal defects have been primarily discovered by studying large families with autosomal dominant forms of septal defects using traditional linkage approaches In vitro experiments demonstrated that the mutant Gata4 protein had a greatly reduced affinity for its binding element with an associated decrease in transcriptional activity and disrupted a novel interaction between Gata4 and Tbx5 GATA4 G296S mutation, and affected members display a similar phenotype of atrial septal defects and incompletely penetrant pulmonary valve stenosis GATA4 is not as well understood.We reported a large pedigree with autosomal dominant congenital heart disease that was associated with a mutation of a highly conserved glycine residue to a serine at codon 296 (G296S) GATA4 G296S mutation in vivo, we generated and characterized transgenic mice that contain the orthologous G295S mutation in the Gata4 murine locus. Here, we show that homozygous Gata4 G295S knock-in (ki) mice (ki/kiGata4 G295S) display normal ventral morphogenesis but early embryonic lethality after the linear heart tube stage. Histologic analysis demonstrates a thin ventricular myocardium in the ki/kiGata4 G295S embryos, which is associated with a cardiomyocyte proliferation defect. Molecular characterization of these mutant embryos demonstrates that expression of Gata4 target genes is decreased in the heart. Echocardiographic examination of heterozygote (ki/wtGata4 G295S) mice found subtle atrial septal defects and semilunar valve stenosis. Embryonic cardiomyocytes from heterozygote mice display cardiac proliferation deficits. Consistent with functional deficits for the Gata4 G295S mutation, compound heterozygote mice harboring only a single Gata4 G295S mutant allele in the early myocardium recapitulated the phenotype seen in ki/kiGata4 G295S embryos. These studies demonstrate the generation of a mouse model for human cardiac malformations and suggest that abnormal cardiomyocyte proliferation may contribute to human atrial and ventricular septal defects caused by mutations in GATA4.To identify the functional deficits of the in vivo functional deficits of the human CHD-causing GATA4 G296S mutation, we generated mice harboring the orthologus mutation at codon 295 in the murine Gata4 gene (G295S). Using a previously published targeting construct, we mutated the nucleotide from G to A resulting in a glycine to serine substitution in exon 3 which contains the C-terminal zinc finger of Gata4 ki) was detected by Southern blotting in chimeric mice and confirmed by direct sequencing in heterozygous and homozygous mice   . The intwt mouse . No evidtermates . Histoloen ovale –S2D alonen ovale –S2F and en ovale –S2H.in vivo functional deficits of the Gata4 G295S mutation, we interbred ki/wtGata4 G295S to generate ki/kiGata4 G295S mice. Analysis at postnatal day 7 demonstrated no ki/kiG295S pups indicating that the homozygous knock-in allele is embryonic or early neonatal lethal. To determine the timing of lethality, we performed timed mating and found that ki/kiGata4 G295S embryos did not survive beyond embryonic day (E)11.5, and normal Mendelian ratios were noted from E8.5–E10.5  by quantitative RT-PCR. The expression levels of all four genes were significantly decreased in ki/kiGata4 G295S mutant hearts as compared to wildtype controls  and Sox17, along with the endoderm transcription factors, Hex1 and Hnf4, that are not direct transcriptional targets of Gata4Afp and Sox17 did not have decreased levels of expression by qRT-PCR in ki/kiGata4 G295S embryos when compared to wildtype littermates at three different embryonic timepoints -based strategy. Hearts were dissected from E11.5 and E13.5 embryos and cardiomyocytes were purified based on cardiac troponin-T expression ki/wtGata4 G295S embryonic atrial and ventricular cardiomyocytes displayed decreased cell proliferation as compared to cardiomyocytes from wildtype littermates at both E11.5 and E13.5 ; αMHC, which is specific for late embryonic myocardium, with robust Cre-mediated excision starting at E9.5; and Nkx2-5, which is expressed in early embryonic myocardium starting at E8.0 and also in the pharynx and liver Cre (enGata4G295S) and αMHC-Cre (lmGata4G295S) showed the expected genotypes were present in normal Mendelian ratios with no evidence of growth retardation  demonstrated partial lethality by E10.5 and evidence of severe growth retardation  and hypoplastic endocardial cushions Gata4 inhibits an interaction with Tbx5 and Gata4-Tbx5 compound heterozygotes display atrioventricular septal defects GATA4 G296S mutation has a role in formation of the endocardial cushions but one family member with the G296S mutation did have an atrioventricular septal defect GATA4 mutations have atrioventricular septal defects Nos3) has been proposed, but the early lethality of the ki/kiGata4 G295S embryos did not allow for analysis of this pathway ki/kiGata4 G295S embryos, the ki/wtGata4 G295S heterozygotes did develop stenosis of the semilunar valves. This phenotype along with recent publications demonstrating bicuspid aortic valve and aortic valve stenosis in Gata5-null and Gata4;Gata5 compound heterozygote mice suggest that endocardial specific deficits of the G296S protein may contribute to the aortic and pulmonary valve stenosis The G295S mutation in Gata4 may result in multiple functional deficits in the embryonic heart. Mice homozygous for the ki/kiGata4 G295S mice demonstrates that the Gata4 G295 mutant allele is not a loss of function allele. Initial human genetic studies demonstrated that haploinsufficiency of GATA4 resulted in cardiac malformations and there was a possibility that the Gata4 G295 was a null allele Gata4 results in a selective loss of some Gata4 functions. Specifically, the Gata4 G295S protein is able to activate downstream target genes in the developing endoderm but not in the cardiac mesoderm. Potential mechanisms for this difference may lie in the inability of Gata4 G295S protein to interact with Tbx5 in the mesoderm to activate downstream targets or that Gata4 may function as a transcriptional co-activator in the endoderm and DNA binding is not necessary. The findings from the conditional deletion crosses, as discussed above, also support that the G295S mutant protein has defined functional deficits in different cell lineages. Additional investigation is needed to identify all the potential functions of Gata4 and then determine which are loss with the Gata4 G295S mutation.The phenotypic analysis of Gata4 G295S mutation results in myocardial hypoplasia due to diminished cardiac proliferation. This phenotype is at least partially mediated by reduced expression of cyclin D2, a member of the D-cyclin family of cell cycle regulators. Mice lacking any single D-cyclin are viable and do not display obvious cardiac defects kiGata4 G295S mice display cardiomyocyte proliferation deficits and suggest that this is a possible mechanism for the atrial septal defects seen in these mice.Cardiomyocyte proliferation is critical for normal cardiac development, and our findings provide evidence that the kiGata4 G295S mice provides a mouse model to study human congenital heart defects. This mouse model will be of significant value to study genetic and environment modifiers for cardiac malformations along with allowing for a more mechanistic understanding of the embryologic basis of septation and valvular defects. While the dosage sensitivity of cardiac transcription factors for normal cardiac morphogenesis is generally well accepted, this mouse model demonstrates that specific mutations may have limited functional deficits. The kiGata4 G295S mice which encodes a partially functional mutant protein, offers us a tool to define these abnormalities in vivo.The generation of the Research was approved by the Institutional Animal Care and Use Committee at University of Texas Southwestern Medical Center (Protocol No. 2008-0094) and Research Institute at Nationwide Children's Hospital (Protocol No. AR09-00040) and conforms to the Guide for the Care and Use of Laboratory Animals.Gata4 G295S mutation. The targeting vector contains an 8.2 kb mouse genomic fragement, which has the 2nd–6th exons of murine Gata4. The construct contains a Neomycin resistance cassette flanked by loxP sites, as a positive selection marker, and HSV-tk, as a negative selection marker. Genomic DNA containing the N-terminal of the zinc finger domain of Gata4 was subcloned into pBluescript II KS (+/−) phagemid (Stratagene). By site-directed mutagenesis, glycine at codon 295 was changed to serine (the codon GGC was changed to AGC). The targeting construct was linearized with PvuI and electroporated into 129SvES cells. Targeted clones were identified by Southern blotting. Six successful targeted ES clones identified by Southern blotting and direct sequencing for the G295S point mutation. Three clones were injected into C57BL/6 blastocysts to generate chimeric mice.A strategy similar to Crispino et al., 2001 was used to generate knock-in mice harboring the Gata4 locus by using fluorescent probes. This method combines PCR and mutation detection in a single step. Two TaqMan  probes were used, one for each allele. This method is implemented using the Applied Biosystems 7500Fast and TaqMan reagents to detect this point mutation in Gata4. The probe and primer sequences are shown in Germline transmission was achieved by mating to C57BL/6 mice. Mice used in this study were on a mixed 129SvEv/C57BL6 genetic background. For genotyping, allelic discrimination assay was used to detect this single nucleotide change in Gata4 G295S mutation were generated and genotyped as described above. Mice heterozygote for Gata4 G295S were mated to generate G295S homozygote embryos. Pregnant mothers were sacrificed at various embryonic timepoints. Littermates were used as controls for histologic sections, gene expression studies and FACS analysis.Mice were maintained on a 0600 to 1800h light–dark cycle, with noon of the day of observation of a vaginal plug defined as E0.5. Mice heterozygous for in vivo ultrasound images were obtained in 8 and 16 week old mice using a VisualSonics Vevo2100 imaging system  with a mechanical transducer (MS400). Mice were anesthetized with isoflourane and echocardiograms were performed in a genotype-blinded fashion. Statistical analysis was performed using Fisher's exact test.Two dimensional and Doppler In situ hybridization was performed as described previously 35S-labeled antisense probes synthesized with T3, T7, or SP6 RNA polymerase  from mouse Hand1, Hand2 and Tbx5 cDNA.For histological analysis, embryos and adult hearts were fixed in 4% paraformaldehyde and paraffin embedded. Hematoxylin and eosin (H and E) staining was carried out on heart sections using standard methodology. t-test.For the immunostaining studies, histologic sections were deparafinnized in xylene and rehydrated in phosphate buffered saline (PBS). Proliferation assays were performed using the phosphohistone H3 (PH3) antibody . The sections were permeabilized in 0.3% Triton X-100 in PBS. Sections were then blocked by 3.5% donkey serum in PBS followed by incubation with 1% rabbit anti-phosphohistone H3 antibody overnight at 4°C. Sections were then washed in PBS and Cy3 (1%) secondary antibodies  for 30 min. For cell proliferation studies, contiguous sections were stained for monoclonal mouse anti-human desmin using Cy3-conjugated antibody  to label the cardiomyocytes. The percentage of PH3-stained ventricular cardiomyocytes/total number of ventricular cardiomyocytes was calculated by analyzing a minimum of three embryos for each genotype. A minimum of four sections per embryo were analyzed, and the means and standard deviations are shown. Apoptosis (TUNEL) assays were performed using the In Situ Cell Death Detection Kit, Fluorescein (Roche) according to manufacturer instructions. Labeled ventricular cardiomyocytes were counted on a minimum of six sections of control and mutant embryonic hearts. Statistical analysis was performed using Student's n = 3 per group. Statistical analysis was performed using Student's t-test, and a p value of less than 0.05 was considered significant.RNA was purified from embryonic hearts (E8.5–E10.5) from mutant embryos and their respective wildtype littermates using Trizol (Invitrogen). Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed using the Taqman Universal PCR Master Mix kit . 100 ng of total RNA was used for reverse transcription and amplification in each real-time PCR reaction using Applied Biosystems 7500 real-time PCR machine. Commercially available SYBR Green (Applied Biosystems) PCR mix was utilized for the following genes: ANF, α-MHC, cTnC, Myl3, CyclinD2, Nkx2-5, Tbx5, Mef2C, β-MHC, HNF4, Hex1, alpha-fetoprotein and Sox17. The sequences for primers are in Gata4 wildtype and Gata4 G295S myc-tagged expression vectors Cyclin D2 pAUG-β-gal reporter vector HeLa cells were transfected using Fugene 6 (Roche) according to manufacturer's instructions with 200 ng of Embryonic heart samples for fluorescence-activated cell sorting (FACS) were prepared in the following manner: 15–20 embryonic hearts of each genotype were dissected, dissociated to a single cell solution, digested with collagenase type II (Worthington) solution, washed, spun down and resuspended in cardiomyocyte staining buffer. Cells were fixed with BD Cytofix/CytopermTM solution, permeabilized and incubated with monoclonal mouse anti-troponin T  and Ki67 . Experiments were performed in triplicate and cells were analysed on a LSRII with DiVa software .Figure S1wt/kiGata4 G295S mice. (A) Scatter plot showing fractional shortening in wildtype and ki/wtGata4 G295S mice at 8 and 16 weeks of age. Scatter plots show (B) left ventricular internal diameter (LVID) at end-diastole, (C) left ventricular internal diameter during systole, (D) left ventricular anterolateral wall (LVAW) thickness at end-diastole, (E) left ventricular anterolateral wall thickness during systole, (F) left ventricular posterior wall (LVPW) thickness at end-diastole, and (G) scatter plot showing left ventricular posterior wall thickness during systole.Summary of M-mode echocardiographic analysis of wildtype and (JPG)Click here for additional data file.Figure S2wt/kiGata4 G295S murine hearts by histologic section. Interatrial communication in the form of patent foramen ovale  is found in wt/kiGata4 G295S mice  as compared to wildtype littermate .  represent high magnification image of boxed area in , respectively. Thickening of aortic valve leaflets is found in wt/kiGata4 G295S mouse (F) that had aortic valve stenosis by echocardiogram as compared to wildtype (E). Thickened pulmonary valve leaflets in wt/kiGata4 G295S mouse are shown (H) as compared to normal leaflets in wildtype littermate (G). RA, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle; AO, aorta; PA, pulmonary artery. Scale bars indicate 200 µm.Patent foramen ovale, aortic valve stenosis and pulmonary valve stenosis in (JPG)Click here for additional data file.Figure S3ki/kiGata4 G295S embryos during development. (A) Normal cardiac looping in wildtype E10.5 embryo as compared to incomplete looping in ki/kiGata4 G295S in E10.5 embryo (B). Red arrow, heart. Scale bars indicate 200 µm.Variable cardiac looping of (JPG)Click here for additional data file.Figure S4ki/kiGata4 G295S embryonic hearts. Quantitative RT-PCR demonstrates no significant change in expression levels of Gata5 and Gata6. in E9.5 ki/kiGata4 G295S hearts when compared to wildtype littermates.Expression of Gata5 and Gata6 is unchanged in (JPG)Click here for additional data file.Figure S5ki/kiGata4 G295S embryos. Coronal sections through E9.5 hearts of wt/kiGata4 G295S (A–D) and ki/kiGata4 G295S embryos (E–H). Radioactive section in situ hybridization demonstrates mRNA expression of Hand1 , Tbx5 , and Hand2  in ki/kiGata4 G295S embryos is similar to wt/kiGata4 G295S littermates. Bright-field images are shown in (A) and (E). A, atria; V, ventricle; OFT, outflow tract.Expression of Hand1, Tbx5 and Hand2 is unchanged in (JPG)Click here for additional data file.Figure S6ki/kiGata4 G295S embryos hearts. Expression of the Gata4 target endoderm genes, (A) alpha-fetoprotein and (B) Sox17 along with expression of (C) Hex1 and (D) HNF4, genes that are not Gata4 targets, is shown in E8.5 (red), E9.5 (green) and E10.5 (black) embryos. Solid bars, wildtype embryos; striped bars, ki/kiGata4 G295S embryos.Expression of endoderm genes is not decreased in (JPG)Click here for additional data file.Figure S7ki/floxGata4 G295S; +Nkx2-5-Cre, ki/floxGata4 G295S; +Tie2-Cre, ki/floxGata4 G295S; α+-MHC-Cre E10.5 embryos. Immunohistochemistry for Gata4 on histologic sections of E10.5 embryos shows decreased myocardial expression (*) and unchanged endocardial expression (arrowhead) in ki/floxGata4 G295S; +Nkx2-5-Cre embryo (B) as compared to wildtype littermate (A). Gata4 expression is decreased in the endocardium (arrowhead) of E10.5 ki/floxGata4 G295S; +Tie2-Cre embryo (D) compared to wildtype littermate (C). Areas of decreased myocardial expression of Gata4 (arrowhead) in ki/floxGata4 G295S; α+-MHC-Cre E10.5 embryos (F) as compared to wildtype littermate (E). Arrowheads, endocardium;V, ventricle; scale bars indicate 200 µm.Expression of Gata4 in (JPG)Click here for additional data file.Table S1Distribution of surviving progeny at postnatal day 10 for the following mouse crosses.(JPG)Click here for additional data file.Table S2Primer sequences.(JPG)Click here for additional data file.


Vitis vinifera L.), several MYB transcription factors have been identified but the interactions with their putative bHLH partners to regulate specific branches of the flavonoid pathway are still poorly understood.Flavonoid pathway is spatially and temporally controlled during plant development and the transcriptional regulation of the structural genes is mostly orchestrated by a ternary protein complex that involves three classes of transcription factors . In grapevine  was assessed in different in vivo systems: yeast, grape cell suspensions, and tobacco. In the first two systems, VvMYB5bL exhibited a modified trans-activation capability. Moreover, using yeast two-hybrid assay, we demonstrated that modification of VvMYB5b transcriptional properties impaired its ability to correctly interact with VvMYC1, a grape bHLH protein. These results were further substantiated by overexpression of R VvMYB5band L VvMYB5bgenes in tobacco. Flowers from L 35S::VvMYB5btransgenic plants showed a distinct phenotype in comparison with R 35S::VvMYB5band the control plants. Finally, significant differences in transcript abundance of flavonoid metabolism genes were observed along with variations in pigments accumulation.In this work, we describe the effects of a single amino acid substitution (R69L) located in the R2 domain of VvMYB5b and predicted to affect the formation of a salt bridge within the protein. The activity of the mutated protein . In addition, this study underlines the importance of an internal salt bridge for protein conformation and thus for the establishment of protein-protein interactions between MYB and bHLH transcription factors. Mechanisms underlying these interactions are discussed and a model is proposed to explain the transcriptional activity of VvMYB5L observed in the tobacco model.Taken together, our findings indicate that VvMYB5b MYB genes constitute a very large family encompassing 198 members in Arabidopsis thaliana for instance. Such large families are also observed in rice (Oryza sativa L.ssp. indica) and grape (Vitis vinifera L.), with no less than 85 and 108 members, respectively Lx2[R/K]x3Lx6Lx3R) with bHLH protein partners . In this study, VvMYB5bNtCHS, NtANS and NtDFR genes in stamens of control plants. This suggests the absence of an efficient regulatory complex in this tissue or the lack of at least one component of the system. However, in corollas of control plants, a baseline expression was detected for the same structural genes on the same control plants supporting the idea of a pre-existing transcriptional network regulating the accumulation of anthocyanins in these floral organs.Overexpression experiments in tobacco suggest the presence of different regulatory mechanisms in stamens and corollas, with regard to flavonoid pathway genes expression. First, none or little expression was observed for the R 35S::VvMYB5btransgenic tobacco stamens, it appears that the presence of the native VvMYB5bR protein and its interaction with endogenous pre-existing protein partner(s) leads to the activation of the entire anthocyanin biosynthetic pathway , and thus not induce the expression of the NtCHS, NtANS and NtDFR genes . Likectively . In the  stamens . These s stamens .L, as previously described for the c-MYB protein [R nor mutated VvMYB5bL were able to bind MBS sequences in EMSA experiments. However, VvMYB5bR did activate the VvCHI promoter when co-expressed with the co-factors AtEGL3 (bHLH) and AtTTG1 (WDR) in grapevine cells  were sterilized in 2.5% potassium hypochlorite, 0.02% Triton X-100 for 10 min, and washed five times with sterile water. After cold treatment at 4°C for 48 h, seeds were germinated on MS medium [in vitro grown plantlets were transferred to soil into individual pots and cultivated in a growth chamber under the same environmental conditions. The suspension culture of grapevine Chardonnay (Vitis vinifera L.) petiole callus was grown in grape Cormier medium as described in [Seeds from wild type and homozygous T2 generation of transgenic tobacco plants  using the SWISS-MODEL server [L server . The obtL server . MisorieL server .VvMYB5b cDNA sequence (gene accession AY899404) used in this study was previously inserted in the pGEM-T-Easy cloning vector  [VvMYB5b using the QuickChange site-directed mutagenesis kit (Stratagene). Reactions were carried out using the following primer pair: 5'-CAAGAGCTGTCGCCTCCTCTGGATGAACTACCTC-3' (sense) and 5'-GAGGTAGTTCATCCAGAGGAGGCGACAGCTCTTG-3' (antisense). The presence of the introduced mutation in the cDNA was confirmed by DNA sequencing. The native R VvMyb5band L VvMYB5bfull length cDNAs were then cloned between the XbaI/SacI restriction sites of the pGiBin19 binary vector between the 35S promoter of the cauliflower mosaic virus and the nopaline synthase (nos) poly(A) addition site, as described in [Agrobacterium tumefaciens LB4404 host strain. Tobacco was transformed and regenerated according to the leaf discs method [The son, WI) . The R69ribed in . Both cos method . SelectiR 35S::VvMYB5band L35S::VvMYB5b. One μg of total RNAs was reverse transcribed with oligo(dT)12-18 in a 20 μl reaction mixture using the Moloney murine leukemia virus (M-MuLV) reverse transcriptase (RT) according to the manufacturer's instructions . Transcript levels of NtCHS, NtF3H and NtDFR endogenous genes and the transgene R/L VvMYB5bwere measured by real-time quantitative RT-PCR, using SYBR Green on an ® iCycler iQ(Bio-Rad) according to the procedure described by the supplier. PCR reactions were performed in triplicate using 0.2 μM of each primer, 5 μl SYBR Green mix (Bio-Rad) and 0.8 μl DNAse treated cDNA in a final volume of 10 μl. Negative controls were included in each run. PCR conditions were: initial denaturation at 95°C for 90 s followed by 40 cycles of 95°C for 30 s, 60°C for 1 min. Amplification was followed by melting curve analysis to check the specificity of each reaction. Data were normalized according to the NtUbiquitin gene expression levels and calculated with a method derived from the algorithms outlined by [EF421430 and EF421429).Total RNA was isolated from wild-type and transgenic tobacco flower tissues according to . At leasR VvMyb5band L VvMyb5bfull length cDNAs were amplified by PCR using the Phusion™ High-Fidelity DNA polymerase (Finnzymes) with oligonucleotides introducing the BamHI (5'-TAATGGATCCATGAGGAATGCATCCTCA-3') and SalI (5'-TAATGTCGACTCAGAACCGCTTATCAGGTTG-3') restriction sites , and cloned in the pDH5 vector , a derivative from the pUC18 vector, that allows constitutive transient expression of the transgene. Integrity of each coding sequence was verified by sequencing  using the pDH5F (5'-CCCACTATCCTTCGCAAG-3') and the pDH5R (5'-CTAATTCCCTTATCTGGGAA-3') primers. Transient co-transfection experiments of grapevine suspension cells and dual-luciferase assays were carried out as previously described in [ribed in .R VvMyb5band L VvMyb5bcDNAs were amplified by PCR using the Phusion™ High-Fidelity DNA polymerase (Finnzymes) using oligonucleotides introducing the BamHI restriction site  at the 5' (5'-TAATGGATCCAGATGAGGAATGCATCCTCA-3') and 3' (5'-TAATGGATCTCAGAACCGCTTATCAGGTTG-3') ends. After enzymatic digestion, PCR fragments were introduced into the pGBKT7 vector , in fusion with the GAL4 DNA Binding Domain (DBD) coding region, under the control of the ADH1 promoter. pGBKT7 vector carries the KanR for selection in E. coli and the TRP1 nutritional marker for yeast selection. The yeast strain AH109 was independently transformed with pGBKT7, pGBKT7-RVvMYB5b, or pGBKT7-L VvMYB5busing the PEG/LiAc method, based on the manufacturer's instructions. Transformants were selected on synthetic dropout (SD) media lacking tryptophan (SD-Trp-) or histidine, adenine, and tryptophan (SD-His-Ade-Trp-). In parallel, positive and negative controls of interaction, provided by the manufacturer, have been performed . As LacZ constitutes the fourth reporter gene of this system, β-galatosidase activity was monitored in recombinant yeasts grown on selective medium. β-galactosidase assays with the ONPG  substrate were performed following the manufacturer instructions. Relative β-galactosidase activity was obtained after normalization with the optical density at 600 nm.R VvMYB5bor L VvMYB5bcoding regions were fused to the GAL4 Activation Domain (AD), and the coding sequence of VvMYC1 was fused to the GAL4-DBD. Because of its intrinsic ability to activate transcription in yeast, VvMYB5bR has not been fused to GAL4-DBD, and consequently the reciprocal combinations were excluded from the two-hybrid assay. R VvMyb5band L VvMyb5bcDNAs were amplified by PCR using the Phusion™ High-Fidelity DNA polymerase with specific primers containing anchors: F, 5'-TTCCACCCAAGCAGTGGTATCAACGCAGAGTGG-3' and R, 5'-GTATCGATGCCCACCCTCTAGAGGCCGAGGCGGCCGACA-3' which allow recombination in the linear plasmid pGADT7-Rec when introduced in yeast. Resulting construct carrying AmpR and LEU selective characteristics allows expression of a GAL4 Activation Domain (AD)-VvMYB5b fusion protein. The VvMYC1 coding sequence (gene accession EU447172) was cloned into the pGBKT7 vector between EcoRI and PstI restriction sites [VvMYC1 construct, R VvMYB5band L VvMyb5bPCR products and the effector plasmid pGADT7-Rec, as described in [-Leu- medium, to make sure that both effector plasmids were indeed integrated to yeast. Transcriptional activation of the reporter gene LacZ was evaluated by monitoring β-galactosidase activity.on sites . The tworibed in . TransfoThe authors declare that they have no competing interests.IH performed most experiments in this paper and wrote the initial manuscript draft. LD cloned VvMYB5bR and VvMYB5bL sequences and transformed tobacco. JB, AM, FR and CTM participated in experiments . BG and TG contributed to analysis and interpretation of structural data. VL, FB and LD conceived the study, participated in the preparation and finalization of the manuscript. EG has revised the manuscript critically for intellectual content. All authors read and approved the final manuscript.IH present address: Groupe de Recherche en Physiologie végétale (GRPV), Earth and Life Institute (ELI), Université catholique de Louvain (UCL), B-1348 Louvain-la-Neuve, Belgium.in vitro synthesized VvMYB5bR/L proteinsDetection of the . Both proteins were produced by the in vitro transcription and translation method with the TnT T7 quick system for the PCR DNA system  according to the manufacturer's instruction. The coding sequences were amplified with Turbo-Pfu (Stratagene) using the following primers pairs: F, 5'-AGATCCTAATACGACTCACTATAGGGAGCCACCATGAGGAATGCATCCTCAGCA and R, 5'-(T)32TCAGAACCGCTTATCAGGTTG. The PCR products were used as template. A 5 μl aliquot of the reagent was used for SDS-PAGE. Separated proteins were transferred onto a nitrocellulose membrane and detected using the Transcend non-radioactive translation detection system . MW in kDa corresponds to the Page ruler prestained #SM0671 protein ladder (Fermentas).Click here for fileAnthocyanin contents in flowers of control and transgenic plants. Anthocyanin pigments were extracted with 1% HCl in methanol in the dark. The anthocyanin concentration is expressed as the absorbance units at 530 nm per gram of fresh tissue weight. Data are the mean of three replicates, and results from two independent transgenic lines are indicated. ND: not detected. Asterisk indicates values that significantly differ from the control .Click here for file


Mammalian target of rapamycin (mTOR)/S6K1 signalling emerges as a critical regulator of aging. Yet, a role of mTOR/S6K1 in aging-associated vascular endothelial dysfunction remains unknown. In this study, we investigated the role of S6K1 in aging-associated endothelial dysfunction and effects of the polyphenol resveratrol on S6K1 in aging endothelial cells. We show here that senescent endothelial cells displayed higher S6K1 activity, increased superoxide production and decreased bioactive nitric oxide (NO) levels than young endothelial cells, which is contributed by eNOS uncoupling. Silencing S6K1 in senescent cells reduced superoxide generation and enhanced NO production. Conversely, over-expression of a constitutively active S6K1 mutant in young endothelial cells mimicked endothelial dysfunction of the senescent cells through eNOS uncoupling and induced premature cellular senescence. Like the mTOR/S6K1 inhibitor rapamycin, resveratrol inhibited S6K1 signalling, resulting in decreased superoxide generation and enhanced NO levels in the senescent cells. Consistent with the data from cultured cells, an enhanced S6K1 activity, increased superoxide generation, and decreased bioactive NO levels associated with eNOS uncoupling were also detected in aortas of old WKY rats (aged 20–24 months) as compared to the young animals (1–3 months). Treatment of aortas of old rats with resveratrol or rapamycin inhibited S6K1 activity, oxidative stress, and improved endothelial NO production. Our data demonstrate a causal role of the hyperactive S6K1 in eNOS uncoupling leading to endothelial dysfunction and vascular aging. Resveratrol improves endothelial function in aging, at least in part, through inhibition of S6K1. Targeting S6K1 may thus represent a novel therapeutic approach for aging-associated vascular disease. Aging is a dominant risk factor for cardiovascular disease Evidence is emerging that mTOR/S6K1 signalling is an important regulator of aging In particular, experiments analysing cardiovascular mTOR/S6K1 activity in aging animal models yield inconsistent results. A study using microarray analyses showed that gene expression pattern associated with mTOR is suppressed upon aging in the heart of Fischer 344 rats It is now well recognized that resveratrol, a natural polyphenol, exerts protective effects on vascular diseases, type II diabetes, and aging in several animal species These findings prompted us to investigate whether S6K1 plays a role in endothelial dysfunction in aging and whether resveratrol protects against endothelial dysfunction through inhibition of S6K1 signalling under the aging condition.To investigate the role of S6K1 in endothelial aging, we first determined S6K1 activity in cultured young and senescent human endothelial cells. The senescence status of the cells was confirmed by higher number of cells which stained positively for senescence-associated ß-galactosidase  in the young cells, as validated by increased S6K1ca protein level and enhanced S6-S235/S236 phosphorylation Fig. 5A,In senescent endothelial cells, the enhanced S6K1 activity was associated with an increased Akt activation, the upstream kinase of mTOR/S6K1, as measured by phosphorylation level of Akt-S473 Fig. 7. Furthermore, the higher superoxide production in the senescent cells was blunted by resveratrol  as well as by rapamycin  Fig. 9C. had much stronger SA-ß-gal staining than that of young rats (1–3 months old) Fig. 10Aen face DHE staining was significantly increased, which was largely reduced by treatment of the aortas with L-NAME  or rapamycin  , Switzerland and was performed in compliance with guidelines on animal experimentation at our institution.trans-resveratrol were purchased from Calbiochem; antibodies against phospho-S6K1-T389 (9205), phospho-S6-S235/236 (2211s), S6 (2217s) were from Cell Signalling ; Antibodies against S6K1 (9205s) was from BD Transduction laboratories ; Alexa Fluor680-conjugated anti-mouse IgG (A21057), dihydroethidium (DHE), MitoSox red mitochondrial superoxide indicator (MitoSox) were from Molecular Probes/Invitrogen , and IRDye800-conjugated anti-rabbit IgG (926–32211) were from LI-COR Biosciences ; the membrane-permeable 4,5-diaminofluoresceine acetate (DAF-2DA) was from VWR international SA ; x-gal was from Promega; Endothelial cell growth supplement (ECGS) pack was from PromoCell GmbH  and all cell culture media and materials were purchased from Gibco BRL .All chemicals including those used for immunoblotting were obtained from Sigma  except the following: l-norepinephrine bitartrate, acetylcholine (ACh), sodium nitroprusside (SNP), en face staining or snap frozen directly in liquid N2 and kept at −80°C till further biochemical analyses.Young (1 to 3 months old) and old (20 to 24 months) male WKY rats (Harlan Netherlands) were maintained on a 12-h light-dark cycle and fed standard chow diet and tap water according to the local guidelines of animal experimentation. The animals were anesthetized with xylazin  and ketamin  and sacrificed. Thoracic aortas were dissected and cleaned from perivascular fat, and subjected to organ chamber experiments, shRNA) and rAd/CMV-HA-S6K1ca (a constitutively active S6K1 mutant F5A-E389-R3A), -HA-m/p-Akt (a constitutively active Akt mutant) and –HA-Akt-KA (a dominant negative Akt mutant) were described previously shRNA and the empty rAd were from Invitrogen life Technologies.rAd expressing shRNA targeting human S6K1 driven by the U6 promoter  overnight at 37°C in a COHuman umbilical vein endothelial cells (HUVEC) Cell or tissue lysate preparation, SDS-PAGE, transfer of SDS gels to an Immobilon-P membranes (Millipore) were performed as previously described Low-temperature SDS-PAGE (LT-PAGE) was performed for detection of SDS-resistant eNOS dimer and monomer as described previously Activity of S6K1 was analyzed by monitoring phosphorylation of its substrate S6 at serine235/serine236 (S235/S236) by immunoblotting.2+-free PBS, and incubated in a modified Krebs-Ringer bicarbonate solution  containing 5 µmol/L of DAF-2DA for 30 minutes. For measurement of superoxide, cells were incubated with 5 µmol/L DHE for 20 minutes or 5 µmol/L MitoSox for 10 minutes for detection of cytoplasmic or mitochondrial superoxide, respectively. For functional eNOS uncoupling study, cells were pre-treated with the eNOS inhibitor L-NAME (1 mmol/L) for 1 hour before DHE was added. The cells were then washed 3 times and images were obtained with Zeiss fluorescence microscopy. The intensity of the fluorescence was quantified by Image J software .For detection of NO, HUVECs were gently washed twice with Ca2 and 5% CO2. After equilibration, resveratrol (10 µmol/L) and rapamycin (20 ng/ml) were added to respective blood vessels for 1 hour. DAF-2DA (5 µmol/L) or MitoSox (5 µmol/L) was then added for 30 minutes or 10 minutes, respectively. For functional eNOS uncoupling study, aortas of young and old rats were treated as above described, except that L-NAME (1 mmol/L) was added to respective blood vessels for 1 hour followed by staining with DHE (5 µmol/L) for 20 minutes. The aortas were then washed three times and fixed in 4% paraformaldehyde followed by counterstaining with DAPI (300 nmol/L for 3 minutes). After washing with Krebs buffer, the aortas were carefully cut longitudinally and mounted en face (face down) on slides and then covered with cover slip for endothelial layer imaging. Vectashield mounting medium was used to preserve the fluorescence. The fluorescence was analyzed in a Leica DM6000 confocal microscope within hours after preparation. Fluorescence from DAF-2DA staining was excited with 488 nm argon laser with emission detection at 500–535 nm, whereas fluorescence from MitoSox was detected at 590 nm emission. Z-scanning was done for each sample. After the signal on the top  of the sample was observed, the images were collected. Three consecutive images per field, acquired through the full thickness of endothelial signal, were recorded for analysis. At least 3 different fields per sample were evaluated. The images from DAF-2DA, MitoSox and DAPI staining were quantified with Image J software and results are presented as the ratio of DAF-2DA and DAPI positive nucleus or ratio of MitoSox and DAPI.NO and mitochondrial superoxide production in the absence or presence of resveratrol or rapamycin was assessed with DAF-2DA and MitoSox staining, respectively. Briefly, young (1–3 months) and old rat (20–24 months) aortas cleaned of perivascular tissues were equilibrated for 30 minutes in Krebs buffer at 37°C aerated with 95% O2 and 5% CO2 at 37°C in a Multi-Myograph System  in parallel. The aortic rings were allowed to equilibrate for 45 minutes and progressively stretched to a passive tension of 10 mN that gives the optimal length–tension relationship. To study the effects of resveratrol or rapamycin on endothelium-dependent or independent relaxations, one ring was incubated with vehicle as control, the others were treated with either resveratrol (10 µmol/L) or rapamycin (20 nmol/L) for 1 hour. The aortic rings were then contracted with norepinephrine (0.3 µmol/L). After the contraction induced by norepinephrine reached plateau, cumulative concentration-responses to acetylcholine  or to the NO donor sodium nitroprusside  were performed in each aortic ring. Indomethacin (1 µmol/L) was added to the aortic rings throughout the whole experiments to prevent any generation of endothelium-derived contracting prostanoids.Aortas were isolated from young and old rats and cleaned of perivascular tissues as described Data are given as mean±SEM. In all experiments, n represents the number of experiments or animals. Statistical analysis was performed with unpaired t test or ANOVA with Dunnett or Bonferroni post-test. For analysis of vascular relaxations to ACh in isolated aortas, relaxation responses to the same concentration of ACh among the different aortic rings were compared. Statistical analysis was performed using ANOVA with Bonferroni post-test. Differences in mean values were considered significant at p<0.05.


Highly pathogenic avian influenza (HPAI) H5N1 virus infection is still a potential threat to public health worldwide. While vaccines and antiviral drugs are currently under development, neutralizing antibodies could offer an alternative strategy to prevent and treat H5N1 virus infection. In the present study, we had developed a humanized antibody against H5N1 viruses from mouse-derived hybridoma in order to minimize its immunogenicity for potential clinical application. The humanized antibody hH5M9 was generated by transferring the mouse complementarity determining region (CDR) residues together with four key framework region (FR) residues onto the FR of the human antibody. This humanized antibody exhibited high affinity and specificity comparable to the parental mouse or chimeric counterpart with broad and strong neutralization activity against all H5N1 clades and subclades except for Egypt clades investigated. Furthermore, through epitope mapping we identified a linear epitope on the top region of hemagglutinin (HA) that was H5N1 specific and conserved. Our results for the first time reported a humanized antibody against H5N1 viruses by CDR grafting method. With the expected lower immunogenicity, this humanized antibody was expected to be more efficacious than murine or human-mouse chimeric antibodies for future application in humans. H5N1 virus, one of highly pathogenic avian influenza (HPAI) strains, has caused numerous outbreaks in poultry in Southeast Asia since 1997 To prevent H5N1 pandemic, world-wide efforts have been made to develop and stockpile preventive vaccines, antiviral drugs as well as passive immune therapies Influenza hemagglutinin (HA), with 16 antigenic distinct subtypes, is the main target for neutralizing antibodies against influenza viral infections Although antibody-based therapy has displayed the potential prevention and treatment of H5N1 virus infection in human, the efficacy of murine mAbs is hampered by their diminished serum half-life, inability to trigger human effector functions and the induction of a human anti-mouse antibody (HAMA) response In the previous study, the mouse monoclonal antibody H5M9 (mH5M9) demonstrated broad and strong neutralizing activity against H5N1 viruses isolated from 1997 to 2006 To generate a human-mouse chimeric antibody, the variable regions of both light chain (VL) and heavy chain (VH) from mH5M9 were subcloned into IgG expression cassette vectors IFH and IFL, respectively. IFH-VH and IFL-VL vectors were co-expressed in 293F cells, yielding chimeric H5M9 (cH5M9) .37, Arg66, Val71, Val109) were retained, which were deduced to influence the conformations of CDRs according to the simulation of three dimensional structure of VH and VL regions  were the most homologous to that of mH5M9, respectively . We thus regions . The rec regions .The amino acid residues of the variable regions of mH5M9, CDR-grafted hH5M9 and FabOX108 antibodies were listed in An indirect ELISA was first used to analyze the antigen-binding activity of cH5M9 and hH5M9 against H5 HA (A/Vietnam/1194/04). As shown in KD) values of mH5M9, cH5M9 and hH5M9 were calculated to be 3.05×10−11, 4.31×10−11 and 3.84×10−10 M, respectively (Ka) of cH5M9, hH5M9 or mH5M9, the dissociation rate constant (Kd) of hH5M9 was slightly faster than other two antibodies, which implied that the substitution of certain residues in antigen-binding sites of VH or VL domains in hH5M9 reduced the binding stability after CDR grafting when compared with its parental or chimeric counterpart.To further quantify the binding ability of the engineering cH5M9 and hH5M9 antibodies, kinetic binding constants were determined by ProteON surface plasmon resonance (SPR) assay using H5 HA (A/Vietnam/1194/04) coated biosensor. According to the association and dissociation curves of antibodies shown in ectively . AlthougIn the previous study, mH5M9 showed broad and strong neutralization activity against H5N1 viruses To further determine the neutralization breadths and potencies of cH5M9 and hH5M9, we had chosen nine H5 HAs sub-classified into clades 0, 1, 2 and 4 for further titration, which had been associated with human or avian pathogenesis to date . cH5M9 aThese result indicated that CDR-grafted hH5M9 might recognize a shared epitope on HAs from almost all clades and subclades of the H5 subtype that was not shared by HAs of H1, H7, and H9 subtypes.22, Asn120, Thr151, Gln152, Asp154, Ile210 and Ser235 were unique to A/Egypt/N05056/09, which inferred these seven amino acids were the candidates for antigenic epitope according to the previous study To anchor the epitope recognized by hH5M9, two fragments of HA, HA1 and HA2, were first prepared and adapted for epitope mapping. Through western blotting analysis, it was demonstrated that hH5M9 antibody reacted with the viral HA1 protein, rather than HA2. Considering the denaturation of HA1 during the western blotting analysis, the epitope targeted by hH5M9 appeared to be linear rather than conformational . AccordiP235G led to the abolishment of the binding ability of HA molecule to hH5M9 whereas other substitutions had no influence on antigen-antibody interaction. These results indicated that Pro235 in HA was critical for the binding to hH5M9.To test the roles of these seven residues on antigen-antibody recognition of the H5 HA protein, we generated seven HA1 mutants based on wild type HA from A/Anhui/1/05 by site-directed mutagenesis. HA1 mutants with single amino acid mutation illustrated in 235 were generated and screened by hH5M9 . To further verify the epitope mapping, we have synthesized the KPNDAINF peptide coupled to KLH and determined the binding ability of hH5M9 to this peptide. The result indicated that hH5M9 interacted with peptide coupled to KLH whereas no response was detectable to KLH only , which fhttp://www.fludb.org/brc/home.do?decorator=influenza) representing major epidemic avian H5N1 genetic clades up to Sep 2012. Among 2376 H5N1 virus strains, 1593 cases contained the epitope sequence identified in this study, indicating that hH5M9 was able to target near 67.0% of H5 strains reported. Moreover, among 243 human H5N1 virus strains, 179 cases contained this epitope with 73.7% coverage  completely restored the binding avidity comparable to its mouse or chimeric counterpart. We thus deduced that the preservation of key mouse FR residues was critical for the successful design of CDR-grafted antibodies probably through constraining the CDR conformations To minimize the immunogenicity of murine antibodies, chimeric and CDR-grafting antibodies were generated. There are two approaches for CDR grafting Although antibody epitopes had been reported to be located in five of the eleven viral proteins of influenza virus, most of the epitopes were located in the viral HA in vitro of hH5M9. The residues Lys234, Pro235, Asn236, Asp237, Ala238, Ile239, Asn240, Phe241 were highly conserved among the 2376 H5N1 virus strains. Interestingly, HAPro235Ser mutation existed in most Egypt clades belonging to clade 2.2.1. When we analyzed the conservation of this epitope in H5N1 strains reported annually, up to 94% of H5N1 strains had “KPNDAINF” before 2005. Inhibition on the viruses was of particular importance due to their wide geographical spread and pathogenicity in human. Based on our study, the antibody hH5M9 might counteract most of them. Meanwhile, similar epitope sequence recognized by hH5M9 in our study was found not to exist in any other HA subtypes. In other words, “KPNDAINF” was an H5N1-specific epitope. The H5 epitope for hH5M9 was also comprised in the H5N1 strain-specific hemagglutinin CD4+ T cell epitope In the present study, hH5M9 was demonstrated to recognize a linear epitope of HA1 , which was located on the top region of HA1 protein. Notably, this epitope was not listed in the reported five antigenic sites A–E in vitro against H5N1 viruses. With the expected lower immunogenicity and the conservation of the epitope recognized by CDR-grafted antibody, we intended to expect that it might exhibit high neutralization breadth and potency in vivo and would be employed as adjunctive treatment against human H5N1 virus infection.In summary, we had generated a CDR-grafted antibody that exhibited high neutralizing properties Mouse monoclonal antibody mH5M9 was elicited by immunization of mice with H5 HA concentrated from H5N1 virus (A/goose/Guangdong/1/96) and characterized as described previously The HA proteins used in this study included: A/goose/Guangdong/1/96 , A/Vietnam/1194/04 , A/Indonesia/5/05 , A/Xinjiang/1/06 , A/Egypt/N05056/09 , A/Anhui/1/05 , A/common magpie/Hong Kong/2256/06 , A/Japanese white-eye/Hong Kong/1038/06 , A/goose/Guiyang/337/06  and A/New Caledonia/20/99 (H1N1) . In addition, inactivated influenza viruses were also used, including A/duck/Anhui/1/06 , A/chicken/Shanxi/2/06 , A/duck/Guangdong/1/96 (H7N3) and A/chicken/Shandong/6/96 (H9N2) .To generate chimeric H5M9 antibody (cH5M9), the VH and VL gene fragments of mH5M9 monoclonal antibody in pMD19-T plasmids were subcloned into IgG expression cassette vectors IFH with the constant region of human antibody heavy chain (IFH-VH) and IFL with the constant region of human antibody κ light chain (IFL-VL), respectively. More detailed information of expression vectors was indicated in To further humanize the mouse-human chimeric antibody, CDR-grafting method was used. We intended to choose human variable regions that were the most homologous to mouse variable regions as the templates for CDR grafting of mH5M9. Sequences of VH and VL domain from mH5M9 were subjected to a BLASTP search against the non-redundant Genbank database, respectively. VH and VL genes from FabOX108 were selected to construct humanized VH and VL regions by substituting CDR regions with mH5M9-derived CDR residues. The antibody structure was explicitly solvated with TIP3P water molecules using VMD The recombinant heavy chain and light chain expression vectors, IFH-VH and IFL-VL, for chimeric or humanized antibodies were co-transfected into 293F cells using FreeStyle™ MAX Reagent  according to the manufacturer's instructions. The culture supernatant was subjected to ELISA assay to determine the expression and the antigen-binding capacity of the engineering antibodies. For purification of antibodies, the culture supernatant was subjected to Protein-A affinity chromatography on a HiTrap™ MabSelect SuRe column . The integrity and purity of chimeric and humanized H5M9 antibodies were assessed by Coomassie blue staining after SDS-PAGE electrophoresis. Protein concentration was determined by RC DC Protein Assay .2O2 were added to each well and incubated for 10 min. The reaction was stopped by the addition of 50 µl 2 M H2SO4, and the optical density was measured at 490 nm by an ELISA plate reader .Indirect ELISA was performed to determine the specificity of antibodies. Briefly, the samples were added to 96-well plates pre-coated with 1 µg H5 HA overnight and incubated at 37°C for 1 h. After washing, 100 µl of horseradish peroxidase- conjugated goat anti-human IgG  or goat anti-mouse IgG  were added to each well and incubated at 37°C for 1 h. Finally, 100 µl of 0.2 M citrate buffer (pH 5.0) containing 0.04% o-phenylenediamine  and 0.03% HIn the experiment for epitope verification, 1 µg of H5 HA from A/Vietnam/1194/04 strain, synthesized KPNDAINF coupled to KLH or KLH only were coated overnight. Diluted hH5M9 antibody were added to the wells and incubated at 37°C for 1 h. Further detection was performed as described above. The peptide containing a C-terminal cysteine for conjugation to KLH was synthesized in Sangon Biotechnology .To determine the biological activity of recombinant hH5M9, the classical HI assays were performed as described elsewhere Surface plasmon resonance (SPR) analysis of hH5M9 was performed on ProteOn surface plasmon resonance biosensor . The H5 HA (A/Vietnam/1194/04) was immobilized on a GLC sensor chip using an amine coupling kit with 946 resonance units (RU) in the testing flow cells. 200 µl of freshly prepared antibodies at various concentrations were injected at a constant flow rate of 100 µl/min with 120 s duration at 25°C. Responses from the HA surface were normalized by that from the mock surface without protein binding or a separate injection with buffer only. An anti-EGFR antibody C225 was used as a negative control. Binding kinetics for the antibodies and the data analysis were performed using ProteON manager software .H5 HA (A/Anhui/1/05) was applied to 12% SDS-PAGE and transferred to a PVDF membrane with a Trans-Blot SD semi-dry transfer cell apparatus . hH5M9 (1 mg/ml) was used as primary antibody. A rabbit polyclonal antibody against A/Anhui/1/05 was used as the positive control. IRDye 800CW-conjugated goat anti-human or goat anti-rabbit IgG  was used as the secondary antibody for visualization. The fluorescent bands were detected by Odyssey Imaging System .22, Ser120, Ile151, Lys152, Asn154, Val210, Thr231, Ile232, Leu233, Lys234, Pro235, Asn236, Asp237, Ala238, Ile239, Asn240, Phe241, Glu242 and Ser243) were site-mutated to glycine (G) by PCR. The site-directed mutant fragments were digested by BamHI and XhoI  and subcloned into pcDNA3.0 vector. All constructs were confirmed by DNA sequencing and subjected to transfection for transient expression of mutant HA molecules in 293T cells. Immunofluorescence assays were subsequently performed to determine the binding ability of mutant HA molecules to hH5M9.Mutagenic primers were designed by software Primer Premier 5.0  after sequences alignment of HA region from H5N1 isolates including A/goose/Guangdong/1/96 (Genbank accession AAD51927), A/duck/Hong Kong/p46/97 (AAF02306), A/Vietnam/1194/04 (ACR48874), A/Indonesia/5/05 (AFM78567), A/Xinjiang/1/06 (ACJ68614), A/Egypt/N05056/09 (ACT15357), A/Anhui/1/05 (ADG59080), A/common magpie/Hong Kong/2256/06 (ABJ96777), A/Japanese white-eye/Hong Kong/1038/06 (ABJ96775) and A/goose/Guiyang/337/06 (ABJ96698). Nineteen amino acid residues in HA gene of A/Anhui/1/05 , and then stained in the wells. Purified hH5M9 (1 mg/ml) was used as primary antibody. A rabbit polyclonal antibody against A/Anhui/1/05 was used as the positive control. Binding antibodies were detected by using Cy2-conjugated goat anti-human or goat anti-rabbit IgG  and observed under immunofluorescence microscope .Figure S1Structure of expression cassette vectors IFH with the constant region of human antibody heavy chain (A) and IFL with the constant region of human antibody κ light chain (B). Variable region of heavy chain was cloned into AflII and NheI sites of IFH vector. Variable region of light chain was cloned into EcoRV and HindIII sites of IFL vector.(TIF)Click here for additional data file.Figure S2Molecular modeling of mH5M9 variable regions. The CDRs were shown in red and the FRs were shown in blue. Eight framework residues, H24, H37, H67, H71, H93, H109, L58 and L87, which were different from FabOX108 while critical for constraining the CDR conformations, were colored in green.(TIF)Click here for additional data file.


BRCA1 and RAD51 genes, which are involved in homologous recombination (HR), a major DSB repair system. siRNA-mediated depletion of CBP and p300 impaired HR activity and downregulated BRCA1 and RAD51 at the protein and mRNA levels. Chromatin immunoprecipitation assays showed that CBP and p300 bind to the promoter regions of the BRCA1 and RAD51 genes, and that depletion of CBP and/or p300 reduces H3 and H4 acetylation and inhibits binding of the transcription factor E2F1 to these promoters. Depletion of CBP and p300 impaired DNA damage-induced phosphorylation and chromatin binding of the single-strand DNA-binding protein RPA following BRCA1-mediated DNA end resection. Consistent with this, subsequent phosphorylation of CHK1 and activation of the G2/M damage checkpoint were also impaired. These results indicate that the HATs CBP and p300 play multiple roles in the activation of the cellular response to DSBs.Histone acetylation at DNA double-strand break (DSB) sites by CBP and p300 histone acetyltransferases (HATs) is critical for the recruitment of DSB repair proteins to chromatin. Here, we show that CBP and p300 HATs also function in DSB repair by transcriptionally activating the  Chromosomal DNA and histones form a highly condensed structure known as chromatin. Access to chromosomal DNA by transcription factors and DNA repair proteins is regulated by chromatin remodeling, including histone modification and nucleosomal alteration BRCA1 and RAD51 genes, which are key regulators of homologous recombination (HR), the other major DSB repair system. In DSB-induced HR, which occurs after DSB recognition by the MRE11-RAD50-NBS1 (MRN) complex, BRCA1 contributes to DNA end resection at DSBs as a member of the CtIP-MRN-BRCA1 complex, allowing the phosphorylation of RPA (RPA70/RPA32/RPA16) and binding of phosphorylated RPA to single-stranded DNA (ssDNA) RAD51 and BRCA1 genes is strictly regulated by E2F transcriptional factors in a cell cycle-dependent manner BRCA1 and RAD51 genes, resulting in the promotion of DSB-induced HR and activation of the DNA damage checkpoint.CBP and p300 are well known to function as transcriptional co-activators by producing “relaxed” chromatin accessible to transcription factors through the acetylation of histones H3 and H4 at gene promoter regions ; however, no previous reports have addressed the contribution of CBP/p300 to the transcriptional activation of genes involved in DSB repair. In fact, we previously showed that the expression levels of genes involved in NHEJ are not significantly affected by CBP or p300 H1299 and A549 human lung cancer cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO2. HeLa and HeLa DR-GFP (kindly gifted from Dr. Komatsu) Camptothecin (CPT) and neocarzinostatin (NCS) were purchased from Sigma. ON-TARGETplus smart pool siRNA duplexes were obtained from Dharmacon. The specific siRNAs used were siNT (non-targeting) (L-001810-10), siCBP (L-003477-00), sip300 (L-003486-00), siBRCA1 (L-003461-00), and siBRCA2 (L-003462-00).Antibodies against the following proteins were used: RAD51 , 53BP1 , RPA32 , phospho-RPA32-pS4/S8 (Bethyl A300-245A-1), RPA70 , Mre11 , RAD50 , NBS1 , H2B , γH2AX , BRCA1 , BRCA2 , ATM , phospho-ATM-S1981 , CHK2 , phospho-CHK2-T68 , CHK1 , phospho-Chk1-S345 , β actin , XRCC4 , DNA-PKcs, , CBP , p300 , E2F1 , E2F4 , KU70 , KU80 , ORC2 , Histone H3 , acetylated H4 (K5/8/12/16) , acetylated H3 (K18) , and acetylated H2B (K5/12/15/20) .5 per well) carrying the DR-GFP reporter were grown in 12-well plates and either transfected with the I-SceI expression plasmid pCBASce (0.5 μg) or mock-transfected. Transfections were performed using Lipofectamine LTX reagent (Invitrogen). Cells were then grown for 48 hr prior to analysis. Cells were harvested by trypsinization, washed with PBS, resuspended in 200 μl PBS, passed through a 40 μm cell strainer, and analyzed using a Guava flow cytometer (Millipore). Flow cytometry profiles were generated using the GuavaSoft 2.2 software (Becton Dickinson). For quantitative PCR analysis, genomic DNA was purified from cells using the DNeasy Blood & Tissue Kit (Qiagen), and 20 ng of DNA was subjected to quantitative PCR to quantify the uncut and repaired DNAs using an ABI 7900HT Fast Real-Time PCR System analyzer (Applied Biosystems). PCR was performed for 40 cycles consisting of denaturation at 95°C for 15 sec, and annealing and extension at 60°C for 60 sec using a Power SYBR Green PCR Master Mix (Applied Biosystems). Primer sets used for quantification of the uncut, repaired, and GAPDH gene body regions are as follows: uncut region , repaired region  and the GAPDH gene body region . For each sample, the proportions of uncut and repaired products were calculated as ratios to the amount of GAPDH gene body region . Uncut product ratios were shown as ratios of the pCBASce plasmid untransfected sample. Repaired product ratios were shown as ratios to the siNT (control siRNA)-treated sample.HeLa cells  paraformaldehyde in PBS for 15 min at room temperature, and then permeabilized with 0.3% (v/v) Triton X-100 for 15 min at room temperature. After fixation, cells were washed three times with PBS and blocked with blocking buffer , 0.1% Triton X-100 in PBS) for 10 min. Cells were incubated with primary antibody (diluted in BB) for 1 hr at room temperature, washed with PBS, and counter-stained for 1 hr at room temperature with Alexa Fluor 488 goat anti-mouse, Alexa Fluor 488 goat anti-rabbit, Alexa Fluor 568 goat anti-mouse, or Alexa Fluor 568 goat anti-rabbit secondary antibodies (Molecular Probes) diluted in BB. Cells were then washed three times with PBS and stained with DAPI (0.4 μg/ml in PBS) to visualize DNA. Coverslips were mounted onto glass slides using Prolong Gold mounting agent (Invitrogen). Confocal images were acquired using an inverted microscope (Carl Zeiss) equipped with a 100× oil lens and the ZEN 2008 software (Carl Zeiss).Cells were harvested, washed with PBS, and lysed in 100 μl NETN300 buffer  containing 1 mM PMSF, a proteinase inhibitor cocktail, and a phosphatase inhibitor cocktail (Roche Diagnostics). Whole cell lysates were mixed with SDS sample buffer. To isolate insoluble chromatin, cells were lysed in NETN300 buffer containing complete protease inhibitor cocktail (Roche), incubated for 30 min on ice, and centrifuged at 4°C for 10 min at 14,000 rpm. The resulting insoluble chromatin was sheared twice by sonication for 15 sec. Lysates were separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with antibodies as indicated. Membranes were blocked overnight with TBS containing 0.1% Tween 20 and 0.5% BSA and probed with primary antibodies. After washing with TBS containing 0.1% Tween 20, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies and visualized using an enhanced chemiluminescence reagent .Cells were treated with the indicated agents and collected by trypsinization 1 hr later. Cells were washed in PBS and then fixed in ice-cold 70% ethanol with gentle vortexing. Fixed cells were re-centrifuged and passed through a 40 μm cell strainer, incubated with PBS containing 200 μg/ml RNase A and 5 μg/ml propidium iodide , and analyzed using a Guava flow cytometer.Cells were plated and transfected with siRNA using Lipofectamine RNAMAX (Invitrogen). At 48 hr after transfection, mRNAs and cDNAs were extracted using the TaqMan Gene Expression Cells-to-CT Kit (Applied Biosystems). Aliquots were subjected to quantitative PCR using gene-specific primer/probe sets  (Applied Biosystems) and an ABI 7900HT Fast Real-Time PCR System (Applied Biosystems). PCR was performed for 45 cycles  using TaqMan Gene Expression Master Mix (Applied Biosystems). For each sample, BRCA1 and RAD51 mRNA levels were normalized against the level of GAPDH mRNA; BRCA1/GAPDH and RAD51/GAPDH ratios were further normalized against the corresponding ratio from the siNT sample.6) were plated in 100 mm dishes and transfected with siRNA using Lipofectamine RNAMAX. At 48 hr after transfection, cells were treated with 1% formaldehyde for 10 min at room temperature to crosslink proteins to DNA, followed by the addition of glycine (0.125 M) to stop the crosslinking. Cells were collected with a cell scraper and washed in ice-cold PBS. The ChIP assay was performed using a ChIP-IT Express Enzymatic kit (Active Motif). Briefly, resuspended cells were subjected to DNA shearing for 10 min at 37°C, followed by the addition of EDTA to stop the reaction. An aliquot of each sample was set aside as the input control, while the remaining portion was subjected to immunoprecipitation at 4°C overnight. The cross-links were then reversed in reverse cross-linking buffer for 15 min at 95°C, and the samples were de-proteinized for 1 hr at 37°C using Proteinase K. Aliquots were subjected to quantitative PCR using gene-specific primer sets  and an ABI 7900HT Fast Real-Time PCR System. PCR was performed for 45 cycles  using Power SYBR Green PCR Master Mix (Applied Biosystems). The enrichment of proteins was measured by two values, normalized and relative ChIP enrichment. The former is used to show the degree of enrichment following a specific pulldown with anti-p300 and anti-CBP antibodies. The latter is used to show the decrease/increase in enrichment following gene knockdown at test and control regions. To calculate normalized ChIP enrichment, first ChIP enrichment at the test sites (the BRCA1 or RAD51 promoters and the GAPDH gene body) was calculated as the fraction of the total input DNA that was pulled down by the specific antibody or the non-specific control IgG. Then, normalized ChIP enrichment at the test sites was calculated by dividing the ChIP enrichment of the specific antibody by that of the non-specific control IgG. The relative ChIP enrichment following siRNA treatment was calculated by dividing the ChIP enrichment at the BRCA1 or RAD51 promoter and the GAPDH gene body in the siRNA-treated sample by the ChIP enrichment at the BRCA1 or RAD51 promoter in the sample treated with non-targeting siRNA (siNT). The antibodies used in the ChIP assay were as follows: CBP (sc-369), p300 (sc-585), E2F1 (sc-251), E2F4 (sc-866), acetylated H4 (K5/8/12/16) (06–866), and acetylated H3 (K18) (07–354).Cells (2×1060Co source) after exposure to the indicated agents for 1 hr. Cells were harvested 1 hr after IR exposure, washed twice with ice-cold PBS, fixed, permeabilized with 70% ethanol, and stored at −20°C for at least 2 hr. Fixed cells were blocked with blocking buffer (0.5% BSA in PBS) for 10 min at room temperature. Cells were then stained with an anti-phospho-Histone H3 (Ser10) antibody at 1∶25 dilution in blocking buffer for 1 hr at room temperature. Washes were carried out three times in PBS. For secondary staining, cells were incubated for 1 hr at room temperature with FITC-conjugated donkey anti-mouse IgG antibody (Jackson ImmunoResearch) diluted 1∶100. After secondary antibody staining, cells were washed three times, incubated with 5 μg/mL PI and 200 μg/mL RNase I in PBS, and analyzed using a Guava flow cytometer. The resulting data were analyzed using the GuavaSoft 2.2 software (Becton Dickinson).Cells were exposed to 3 Gy ionizing radiation (IR) . Cells were grown for 5 hr before treatment with CPT. Following a 14 day recovery period, cells were fixed in 50% (v/v) methanol/0.01% (w/v) crystal violet solution for 5 min. Survival points were assayed in triplicate. The number of colonies derived from treated cells was normalized against the number of colonies derived from untreated cells.in vivo by using a GFP-based chromosomal assay in which a DSB is generated by expression of the I-SceI endonuclease, whose recognition site is integrated in the GFP gene such that digestion disrupts the gene . The proportion of repaired products, but not that of the cut products, was reduced after ablation of CBP and/or p300 , but not of γH2AX and 53BP1 foci .CBP and p300 function as transcriptional activators by acetylating histones at gene promoter regions BRCA1 and RAD51 mRNA levels decreased upon CBP/p300 knockdown, suggesting that CBP and p300 act as transcriptional co-activators for these two genes (H1299 cells in  and HeLa and A549 cells in S2C–F). RAD51 and BRCA1 transcription levels are lowest during G0/G1 and highest in the S and G2 phases of the cell cycle RAD51 and BRCA1 transcripts were due to changes in the cell cycle profile, cell cycle progression was examined after depletion of CBP and p300. Knockdown of either of these genes did not affect cell cycle profiles . As in the case of BRCA1 depletion, knockdown of CBP and p300 also decreased the levels of RPA32 and RPA70 proteins in the chromatin fraction of CPT-treated cells, indicating that their recruitment to damaged chromatin had been impaired . The formation of CPT- and IR-induced BRCA1 foci was also impaired upon depletion of CBP and p300 (H1299 cells in S3D–E). Thus, knockdown of CBP and p300 results in BRCA1 deficiency, which in turn reduces the generation of ssDNA.BRCA1 forms a complex with CtIP and MRE11/RAD50/NBS1, which promotes DSB end resection to generate RPA-coated ssDNA and FigureS4A–B). These results indicate that the depletion of CBP and p300 impairs not only HR but also the DNA damage checkpoint by suppressing ATR-CHK1 signaling. This result is supported by the observation that CBP- and p300-depleted cells are more sensitive than control cells to CPT (4C–D).The BRCA1-CtIP-MRN complex promotes the processing of DSBs to generate RPA-coated ssDNA, which is required for the activation of ATR BRCA1 and RAD51, two key genes involved in HR, through acetylation of histones H3 and H4 at the promoter regions of these two genes. These results suggest that DSB-induced HR is regulated by the HAT activities of CBP and p300 in two ways: histone acetylation at promoter regions of HR-related genes, which up-regulates and maintains HR protein expression, and histone acetylation at DSB sites, which allows recruitment of proteins required for HR repair. By functioning as co-activators, CBP and p300 act as hubs in several gene regulatory networks and thereby regulate a variety of intracellular processes BRCA1 and RAD51 in HR contrasts with the regulation of factors involved in NHEJ, the other major DSB repair system, in which CBP and p300 are not involved in transcriptional activation of key genes Previously, we showed that CBP and p300 are recruited to DSB sites, where they acetylate histones H3 and H4 BRCA1 and RAD51 are transcriptionally regulated by CBP and p300. BRCA1 has multiple functions in several DNA repair pathways and the DNA damage checkpoint, whereas RAD51 exclusively functions in HR BRCA1 and RAD51 genes, may be important for both DSB repair and the DNA damage checkpoint. One of the functions of the BRCA1 protein is activation of a DNA end resection complex. Indeed, in this study, we showed that downregulation of BRCA1 upon depletion of CBP and p300 impairs the generation of ssDNA essential for RPA binding, leading to abrogation of HR and ATR-CHK1 signaling. Depletion of CBP and p300 impaired the activation of the G2/M DNA damage checkpoint associated with suppression of ATR-CHK1 signaling. On the other hand, CBP and p300 also contribute to the intra-S checkpoint through their interactions with ATR CHK1 expression. Further studies are needed to elucidate the mechanisms by which the DNA damage checkpoint is regulated by CBP and p300.We have shown here that RAD51 and BRCA1 genes. Notably, CBP and p300 have also been shown to be critical for cell proliferation CBP and p300 proteins acetylate histones and non-histone proteins by interacting with hundreds of proteins and thereby regulate diverse intra-nuclear processes Figure S1Involvement of CBP and p300 in HR. (A) Suppression of I-SceI-induced HR repair upon CBP and p300 depletion. Representative flow cytometry images for the experiments in B) Immunoblots of whole cell lysates of cells harvested 48 hr after transfection with the indicated siRNAs. (C) CPT-induced formation of RAD51 foci. A549 cells were transfected with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr, treated with DMSO (NT) or 1 μM camptothecin (CPT) for 4 hr, and then analyzed by immunofluorescence. The percentages of cells with >10 RAD51 foci are shown. Data represent the mean ± SD.  IR-induced formation of RAD51 foci. H1299 or HeLa cells were transfected with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr, treated with 10 Gy ionizing irradiation (IR) for 4 hr or not irradiated (NT), and analyzed by immunofluorescence. Percentages of cells with >10 RAD51 foci are shown. Data represent the mean ± SD.(TIF)Click here for additional data file.Figure S2Cell cycle profiles after CBP and p300 depletion.  Down-regulation of BRCA1 and RAD51 proteins upon depletion of CBP and/or p300. Cells  were transfected with non-targeting (siNT) or CBP+p300 (siC+p) siRNAs for 48 hr and analyzed by immunoblotting. (C–F) Reduction of BRCA1 and RAD51 transcripts in CBP- and p300-depleted cells. Cells  were transfected with non-targeting (siNT) or CBP+p300 (siC+p) siRNAs for 48 hr and analyzed by quantitative real-time PCR for the detection of BRCA1  and RAD51  mRNAs. Expression levels were normalized against the levels of GAPDH mRNA. Data represent the mean ± SD.(TIF)Click here for additional data file.Figure S3Impairment of RPA foci formation and checkpoint activation in CBP and p300 depleted cells. (A) Effects of CBP and p300 depletion on DSB-induced RPA and CHK1 activation. HeLa cells were transfected with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr, treated with DMSO (NT) or 500 ng/ml neocarzinostatin (NCS) for 2 hr, and whole cell extracts were subjected to immunoblot analysis.  Decreased DSB-induced RPA32 foci formation. HeLa cells were transfected with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr, treated with DMSO (NT) or 1 μM camptothecin (CPT) for 3 hr, and subjected to immunofluorescence analysis. The percentages of cells with >10 RPA32 foci (B) or phosphorylated RPA32 (pS4/S8) foci (C) are shown. Data represent the mean ± SD.  CPT- and IR-induced formation of BRCA1 foci. HeLa cells were transfected with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr, treated with 1 μM camptothecin (CPT) for 3 hr (D) or 10 Gy ionizing irradiation (IR) for 4 hr (E), and then subjected to immunofluorescence analysis. The percentages of cells with >10 BRCA1 foci are shown. Data represent the mean ± SD.(TIF)Click here for additional data file.Figure S4Impairment of DNA damage checkpoint activation and sensitivity to camptothecin in CBP and p300 depleted cells.  Suppression of the G2/M DNA damage checkpoint. HeLa (A) or A549 (B) cells were transfected with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr. The cells were then irradiated  or not irradiated (No IR) and, after 1 hr, they were stained with anti-phospho-histone H3 antibody and propidium iodide (PI). The percentage of mitotic cells (mitotic index) was determined by flow cytometry. Data represent the mean ± SD.  Colony formation of HeLa or A549 cells treated with CPT. HeLa (C) or A549 (D) cells were transfected with non-targeting (siNT) or CBP and p300 (siC+p) siRNA for 48 hr. siRNA-treated cells were replated and incubated for 10–14 days. Survival is expressed as a percentage representing the number of colonies in treated samples relative to the number in DMSO-treated samples. Data represent the mean ± SD.(TIF)Click here for additional data file.


In response to insulin, glucose transporter GLUT4 translocates from intracellular compartments towards the plasma membrane where it enhances cellular glucose uptake. Here, we show that sera from various species contain a factor that dose-dependently induces GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes, human adipocytes, myoblasts and myotubes. Notably, the effect of this factor on GLUT4 is fully maintained in insulin-resistant cells. Our studies demonstrate that the serum-induced increase in cell surface GLUT4 levels is not due to inhibition of its internalization and is not mediated by insulin, PDGF, IGF-1, or HGF. Similarly to insulin, serum also augments cell surface levels of GLUT1 and TfR. Remarkably, the acute effect of serum on GLUT4 is largely additive to that of insulin, while it also sensitizes the cells to insulin. In accordance with these findings, serum does not appear to activate the same repertoire of downstream signaling molecules that are implicated in insulin-induced GLUT4 translocation. We conclude that in addition to insulin, at least one other biological proteinaceous factor exists that contributes to GLUT4 regulation and still functions in insulin resistance. The challenge now is to identify this factor. GLUT4 is the principle glucose transporter that is responsible for the insulin-induced uptake of glucose by muscle and adipose tissue after a meal. The main characteristic feature of GLUT4 is the absence of cell surface recycling in non-stimulated cells O-GlcNAc modification of proteins is linked to insulin resistance In insulin resistance, a condition related to type 2 diabetes, insulin no longer leads to an efficient translocation of GLUT4 towards the cell surface. Insulin resistance has been associated with a number of cellular phenomena which are likely to be linked. First, the level of reactive oxygen species (ROS) is increased under conditions of insulin resistance, while antioxidants are able to ameliorate insulin sensitivity and glucose uptake Taken together, multiple cellular mechanisms contribute to the development of insulin resistance and the associated reduction in GLUT4-mediated glucose uptake. It would be therapeutically relevant to discover novel ways to increase cell surface GLUT4 levels. Here, we present evidence for the existence of a putative factor in serum that displays an insulin-like effect regarding GLUT4 translocation and cellular glucose uptake. Remarkably, the effect of this factor on GLUT4 is additive to the action of insulin and is fully maintained in insulin resistance.3T3-L1 preadipocytes were obtained from ATCC/LGC Standards . Plat-E cells were generously provided by Dr Toshio Kitamura . Bovine and human sera were from PAA . Rabbit serum was withdrawn from New Zealand rabbits. Media and HEPES were from Invitrogen , insulin from Lilly , PDGF-BB, IGF-1, and HGF from PeproTech , and 2-[3H]deoxyglucose from PerkinElmer Life . Monoclonal anti-HA antibody was from Covance , mouse IgG antibody from Sigma-Aldrich , polyclonal GLUT4, GLUT1, and insulin receptor (IRβ) antibodies and HRP-conjugated secondary antibodies from Santa Cruz , antibodies against PAS, ERK, phospho-ERK, Akt, phospho-Akt (T308 and S473), AMPK, phospho-AMPK, and phosphotyrosine from Cell Signaling Technology , anti-AS160 from AbCam , and anti-phospho-AS160, anti-TfR and fluorescent antibodies from Invitrogen. IRS-1 antibody has been described before 5′-GATCGACTAGGGTCCATAGATACGGAGAATCAATATTACCAGAGATCGATTATCCGTACGATGTTCCTGATTATGCTGAGACCACGCTCACCACGCTCTGG and 3′-GATCGTCGACCTCGAGTCACACTTGGGAATCAGCCCC. The sequence of the PCR parts was verified by sequence analysis (Cogenics/Beckman Coulter).The cDNAs encoding HA-GLUT4  and HA-TfR (TfR with an HA tag at its C-terminus) inserted in pBABE-puro vector have been described elsewhere Preadipocytes were cultured and differentiated as described before 2O2. After 15 minutes, the pervanadate was used at a concentration of 100 µM Pervanadate was freshly prepared by combining 639 µl PBS, 300 µl 100 mM sodium vanadate and 61 µl 3% HFor the induction of insulin resistance, a previously described procedure was used Human adipocytes were cultured and differentiated as described elsewhere The fluorescence-based assay for the detection of cell surface GLUT4 levels and the method to measure GLUT4 internalization have been described previously 4, 1 mM CaCl2, 0.4 mM NaH2PO4, 0.6 mM Na2HPO4). Cells were treated or not with insulin or serum for 20 minutes. To control cells, 50 µM cytochalasin B was added (assay background). Cells were incubated for 3 minutes with 0.1 mM 2-[3H]deoxyglucose (0.28 µCi/well), extensively washed with ice-cold phosphate-buffered saline (PBS), and lysed in 1% Triton X-100. Radioactivity was measured by scintillation counting. To be able to compare serum-induced with insulin-induced 2-DOG uptake, either unlabeled glucose was added to the insulin incubations  was fractionated on a 24 ml Sephacryl 200-HR column using an elution buffer consisting of 150 mM NaCl and 10 mM tris pH 7.4 and a flow rate of 5.0 ml/hr. Calibration markers blue dextran , amylase (200 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (67 kDa), ovalbumine (43 kDa), chymotrypsinogen (26 kDa), RNase A (14 kDa) and 2-[3H]deoxyglucose (0.2 kDa) were fractionated using identical volumes and flow rates. Serum fractions (0.75 ml) were concentrated 5 times using 3K Amicon Ultra concentrators before subjecting to the fluorescence-based GLUT4 assay described above.t-tests and comparisons between dose-response curves were evaluated using nonlinear four-parameter sigmoidal dose - response curve fittings and F-tests (Graphpad Prism software). Differences between data sets were considered statistically different when P<0.05.All data are presented as average ± SD. Experiments were performed at least three times. Representative experiments are shown. Comparisons between data sets were evaluated using two-tailed Student's Insulin is the master regulator of glucose homeostasis via its action on intracellular GLUT4 traffic. To test whether there exist other factors in serum that regulate cell surface GLUT4 levels, 3T3-L1 adipocytes were incubated for 20 minutes with 100 nM insulin, 50% fetal bovine serum (FBS), or left untreated. Cells were subsequently immunolabeled with anti-GLUT4 antibody, and analyzed by microscopy . We obseNext, we measured GLUT4 translocation quantitatively, using an assay that is based on the culture and labeling of HA-GLUT4-expressing adipocytes in 96 well plates To establish whether the effect of FBS on GLUT4 was due to an increase in exocytosis or to a reduction in internalization, GLUT4 internalization was measured . In brieTo determine whether the GLUT4-translocating activity of serum was due to the presence of factors that have previously been shown to alter cell surface GLUT4 levels or glucose transport under certain conditions, we tested platelet-derived growth factor PDGF; , insulinTo determine whether the translocation-inducing serum factor was exclusively present in FBS, we also studied newborn bovine, adult bovine, rabbit and human serum . We obsein vitro differentiated human adipocytes, FBS and human serum induced an insulin-independent increase in glucose uptake . The effect of FBS on GLUT4 was inhibited more than 50% by inhibitors of PI 3-kinase as well as by staurosporine, 24 hour herbimycin A treatment, Akti-1/2, and U73122, indicating the involvement of PI 3-kinase, a serine/threonine protein kinase, and phospholipase C. Although serum-induced GLUT4 translocation was inhibited by Akti-1/2, serum did not induce phosphorylation of Akt2 nor of its downstream effector AS160 r 33 and 170 kDa), while it is not retained on a hydrophobic LH20 column (not shown). This may imply that two proteins are involved. Alternatively, the implicated factor may in part be associated with other proteins. Interestingly, heating FBS for up to 10 minutes at 100°C precipitates 75% of the protein content, while leaving the serum GLUT4-translocating activity intact (not shown), implying thermostability and suggesting that it is not the mere presence of a bulk of protein that induces GLUT4 translocation but that the effect is more specific. This is also evident from our size exclusion chromatography studies, in which GLUT4-translocating activity is present in fractions that contain minimal amounts of protein.At present it is unclear what is the nature of the serum factor(s). The factor is likely to be a protein as the GLUT4-translocating activity is collected in the protein fraction upon ammonium sulfate precipitation (data not shown). Moreover, on a size exclusion column, it fractionates into two major peaks physiological relevance. Unfortunately, the activity-based purification of unidentified proteins from serum has proven often to be a difficult and laborious task.This factor may be regulated in a way similar to insulin in that its serum levels may oscillate depending on food intake. Moreover, its serum levels may be altered in insulin resistance in vitro and in vivo.Taken together, we have demonstrated the presence of a factor  in serum that induce GLUT4 translocation in a manner that is largely independent of insulin and insulin signaling and whose effects are fully maintained in insulin resistance. Hence, this factor may prove to have beneficial effects in type 2 diabetes. Now, the challenging task is to identify this factor and to examine its effects on GLUT4 and glucose uptake under normal conditions and conditions of insulin resistance Table S1Insulin concentrations in the sera used in the described studies.(TIF)Click here for additional data file.Figure S1The relative effect of serum on cellular glucose uptake is lower than its effect on cell surface GLUT4 levels. FBS was extensively dialyzed against KRP buffer (cut-off 3 kDa). 3T3-L1 adipocytes expressing HA-GLUT4 (A) or not expressing HA-GLUT4 (B) were serum starved for 2 h in DMEM containing 0.2% BSA, washed with KRP, and incubated for the indicated time periods in the absence (open triangles) or presence of 100 nM insulin (open squares), 25% FBS (filled triangles), 50% FBS (filled squares), or 100% FBS (filled diamonds) in KRP. Subsequently, (A) non-permeabilized fixed cells were immunolabeled with anti-HA and fluorescent secondary antibodies followed by measurement of the fluorescence (arbitrary units) or (B) the uptake of radiolabeled 2DOG during a 2 min incubation was measured. The dashed line in the panels represents basal cell surface GLUT4 levels/2DOG uptake.(TIF)Click here for additional data file.Figure S2Serum does not reduce GLUT4 internalization. 3T3-L1 adipocytes expressing HA-GLUT4 were incubated for 20 min with 100 nM insulin, followed by cooling down on ice, removal of insulin, and cell surface labeling of the cells with anti-HA antibody. Excess antibody was removed, cells were transferred to 37°C, and fixed after various time periods. Permeabilized cells were immunolabeled with fluorescent goat-anti-mouse antibody and analyzed by fluorescence microscopy. Cells were scored for the presence of internalized anti-HA label. Hyperosmolarity (0.45 M sucrose) was included as positive control.(TIF)Click here for additional data file.Figure S3The effect of FBS on GLUT4 is not mediated by PDGF, IGF-1, or HGF. (A) Adipocytes were incubated for 20 minutes with various concentrations of the indicated ligands and relative cell surface GLUT4 levels were determined. (B) 3T3-L1 adipocytes and preadipocytes were incubated for 5 minutes with 100 nM insulin, 2 nM PDGF-BB, 25 nM IGF-1, or 2.6 nM HGF, and lysate samples were subjected to SDS-PAGE and immunoblotting using phospho-ERK and ERK antibodies.(TIF)Click here for additional data file.Figure S4HA-tagged GLUT1 is correctly localized. 3T3-L1 adipocytes expressing GLUT1 with an HA-epitope tag in its first extracellular domain were immunolabeled using anti-HA and anti-syntaxin 13 antibodies. Control adipocytes were immunolabeled using anti-GLUT1 and anti-syntaxin 13 antibodies. Note that the localization of HA-GLUT1 is similar compared with endogenous GLUT1. Bar, 5 µm.(TIF)Click here for additional data file.