Hepatocyte nuclear factor 4α (HNF4α) is a transcription factor that acts as a master regulator of genes for several endoderm-derived tissues, including the intestine, in which it plays a central role during development and tumorigenesis. To better define the mechanisms by which HNF4α can influence these processes, we identified proteins interacting with HNF4α using stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomics with either immunoprecipitation of green fluorescent protein (GFP) or with proximity-dependent purification by the biotin ligase BirA (BioID), both fused to HNF4α. Surprisingly, these analyses identified a significant enrichment of proteins characterized with a role in DNA repair, a so far unidentified biological feature of this transcription factor. Several of these proteins including PARP1, RAD50, and DNA-PKcs were confirmed to interact with HNF4α in colorectal cancer cell lines. Following DNA damage, HNF4α was able to increase cell viability in colorectal cancer cells. Overall, these observations identify a potential role for this transcription factor during the DNA damage response. The transcription factor hepatocyte nuclear factor 4α (HNF4α) regulates the development and differentiation of epithelia in digestive and accessory digestive organs ,2,3. WitAn aberrant expression of the isoform classes of HNF4α seems to be implicated in oncogenesis. The HNF4α expression is altered in cancers of all the tissues where it is expressed. More specifically, an increase in the P2-HNF4α expression coincides with a cancerous state .For colorectal cancer, and most other cancers, an abnormal DNA repair response underlies the initiation and progression of oncogenesis. Colorectal cancer features genomic instability that can typically be categorized into three subtypes: Chromosomal instability (CIN), microsatellite instability (MSI), and CpG-island methylator phenotype (CIMP) . These gIn this study, we sought to investigate the roles played by the P2-HNF4α isoform class in colorectal cancer by identifying its interactome. Using SILAC-based quantitative proteomics approaches , we idenHNF4A can produce two classes of isoforms from two unique promoters, designated P1 and P2 . Th. ThVIL1 p = 2.3 × 10−6), DNA binding (p = 1.8 × 10−5) and DNA repair (p = 1.8 × 10−5) (VIL1 and HNF1A gene transcripts in HEK293T cells (p = 7.5 × 10−33), Chromatin regulators (p = 4 × 10−32), Cell cycle (p = 1.2 × 10−8) and DNA repair (p = 3.2 × 10−7) as the most significant biological functions associated with the protein pulldowns (An in vitro affinity capture assay coupled to SILAC quantification of interacting proteins was next designed in HEK293T cells with the use of the HNF4α7-eGFP recombinant protein as bait (heavy) and eGFP alone used as a control (light) to subtract non-specific interactions A. P2-GFP × 10−5) C. Intere × 10−5) B, have n × 10−5) A. P2-HNF3T cells . Endogen3T cells . The genulldowns C. A compulldowns D. Analysulldowns E. To further explore the relevance of the capacity of HNF4α to recruit the DNA damage response protein partners in colorectal cancer, HCT116 cells that are devoid of the HNF4α expression  were useTo further test whether the DNA binding domain (DBD) or the AF-2 transactivation domain of P2-GFP was necessary for its interaction with DNA repair proteins, various mutant constructs within the DBD and the AF-2 region were generated A. None oTo further evaluate whether HNF4α played a functional role during the DNA damage response, we generated a stable HCT116 cell line for P2-GFP using the Flp-In T-REx system allowing genome integration at a single and known location under the control of a doxycycline-inducible CMV promoter. Immunofluorescence microscopy performed on cells incubated or not with doxycycline confirmed induced nuclear expression of P2-GFP under these conditions A. HCT116HNF4α expression has been shown to be crucial for the survival of CRC cell lines in culture and during intestinal polyposis in mice ,12,37. RDNA repair is not a role traditionally ascribed to HNF4α or more generally speaking, to other tissue-specific master gene regulators. Recent findings have however identified specific transcription factors to act as DNA repair entities independently of their classical transcriptional role when recruited at DNA damage sites . One of Our analysis showed that P2-HNF4α interacts with PARP1, a crucial component of various pathways for the DNA repair . In addi2. For the SILAC culture, cells were incubated in Light or Heavy media for at least four passages at a sub-cultivation ratio of 1:3 to 1:5 in order to fully incorporate isotopically labeled amino acids into their proteomes. Light media was composed of L-Arginine R0 and L-Lysine K0  and Heavy media of L-Arginine R10 and L-Lysine K8 . SILAC media were composed of DMEM  containing supplements with 75 µg/mL of L-Arginine, 113 µg/mL of L-Lysine, 10% triple dialyzed fetal bovine serum (Gibco), 1× GlutaMAX , 10 mM HEPES and 100 U/mL penicillin/streptomycin.The HEK293T human fetal kidney transformed cell line and colorectal cancer cell lines HCT116, HT-29 and LoVo were obtained from the American Type Culture Collection . HEK293T cells were cultured in the DMEM medium, HCT116 cells in McCoy’s medium while LoVo cells in the F-12K medium. All culture media were supplemented with 10% fetal bovine serum , 10 mM HEPES, and 1× GlutaMAX . Cells were maintained at 37 °C in a humidified atmosphere containing 5% COBamHI and EcoRV sites of the in-house modified pLenti6/V5 vector with the addition of a Kozak sequence. Mutated P2-GFP constructs were generated through the PCR site-directed mutagenesis using primer pairs  was then amplified by PCR from the pGLAP5-BirA vector [SpeI and SacII restriction sites. Stable cell lines were generated by infection with lentivirus particles and selection with the use of 8 µg/mL of blasticidin. To generate a doxycycline inducible P2-GFP HCT116 cell line, the HNF4α7 sequence was amplified by PCR, inserted into the pENTR11 vector and then further transferred into the pgLAP5.2-GFP vector [The P2-GFP recombinant lentiviral expression vector was synthesized and subcloned into the pLenti6/V5 vector  by Feldan Inc. . Briefly, an expression-optimized DNA sequence based on the mRNA of HNF4α7 isoforms (NM_001030003.1) and fused at its 3′ end to the eGFP sequence (AF188479) with the linker sequence TTCACTAGTTCAGAATTC was synthesized. This DNA fragment was inserted between the er pairs . BrieflyA vector  and inseP vector  by GatewTCqT-CqT(sampl) × ERCqR(sampl)-CqR where ET/ER and CqT/CqR are the reaction efficiency (E) for quantification cycle (Cq) of the target (T) and reference (R) gene for both the experimental sample (sampl) and the calibrator sample .Total RNA from cells was extracted using the QIAGEN RNeasy kit and subjected to DNAse treatment . RNA of 2 µg quantified by NanoDrop 2000 (Thermo Scientific) were used for reverse transcription with AMV-RT (Roche) and synthesized cDNAs diluted to 5 ng/µL prior to the qPCR analysis. qPCR reactions were done using a Lightcycler 96 apparatus (Roche) in a total volume of 20 µL containing 10 µL of FastStart Essential DNA Green Master (Roche), 500 mM of each primer and 10 ng of cDNAs . Amplico6 of HCT116 cells were seeded in 60 mm dishes and transfected with 14 µg of pDEST47-HNF4α mutant vectors using Lipofectamine 2000 (Thermo Fisher Scientific) before total protein was extracted 48 h later as described above. Protein extracts of 400 µg were then incubated overnight with 20 µL of GFP-Trap beads  or control protein-A sepharose CL-4B beads  before being washed six times in the Triton buffer and eluted 10 min at 95 °C in 20 µL NuPAGE LDS Sample Buffer 1×. Immunoblots were performed using 2 µg or 40 µg of total protein extracts or 20% to 50% of the immunoprecipitated samples and immunoprecipitation validated with an anti-GFP .Total protein extracts from LoVo and HT-29 cells were prepared using the Triton buffer  followed by three rounds of 10 s sonication at 20% (Fisher FB-120 Sonic Dismembrator) to completely lyse nuclei. Immunoprecipitations were performed with an overnight incubation of 400 µg of protein extracts and 4 µg of anti-HNF4α  or anti-DNA-PKcs  followed by a two-hour incubation of 20 µL of MagnaChIP Protein G beads . Co-immunoprecipitated proteins were then eluted from the isolated beads by a 10 min incubation at 95 °C in NuPAGE LDS Sample Buffer 1× (Invitrogen-Thermo Fisher Scientific). Immunoblots were done using the NuPAGE Bis-Tris 4–12% gel and the XCell SureLock system (Invitrogen-Thermo Fisher Scientific) using 2 µg or 40 µg of total protein extracts or 50% to 100% of the immunoprecipitated samples. Anti-GFP (Santa Cruz Biotechnology), anti-PARP-1 , anti-DNA-PKcs , anti-RAD50 , anti-βactin  and several anti-HNF4α   were used for immunoblotting. For the immunoprecipitations of HNF4α mutant constructs, 3.5 × 102, 10 mM KCl, 0.5 mM DTT) and dounced-homogenized 25 times with a tight pestle to isolate nuclei. Nuclei were recovered by centrifugation and further purified on a sucrose gradient. Clean nuclei pellets were resuspended in RIPA 1× buffer  and sonicated five times for 10 s at 25% on a Fisher FB-120 Sonic Dismembrator with a 1/8-inch probe (Thermo Fisher Scientific). Nuclear extracts were further diluted in RIPA 1× buffer and incubated one hour at 4 °C with 40 µl of GFP-TRAP beads  to immunoprecipitate the P2-HNF4α-eGFP protein and interacting partners. Beads were washed three times with RIPA 1× and two times with PBS 1× buffer. After the last wash, beads from the different conditions were pooled together before recovering the proteins by heating the beads at 95 °C for 10 min in NuPAGE LDS Sample Buffer 1× (Invitrogen-Thermo Fisher Scientific). At all steps, a protease inhibitor cocktail  was added to avoid protein degradation. The recovered proteins were then analyzed by mass spectrometry after being reduced to peptides by in-gel digestion as previously described [HEK293T cells were infected with P2-HNF4α-eGFP or empty control lentiviruses and selected with 8 µg/mL of blasticidin for 5 to 10 days until all cells in an uninfected control Petri dish were killed by the selection. In order to perform quantitative mass spectrometry, the proteome of P2-HNF4α-eGFP and empty control cells were labelled using the SILAC technique by successive amplification in heavy and light media, respectively. To perform the GFP-TRAP assay, two Petri dishes of 150 mm were used as starting material for each condition. Cells were recovered using trypsin and washed twice carefully with D-PBS 1×. Nuclear protein extracts were prepared as previously described . Brieflyescribed . HEK293T and HCT116 cells were infected with lentiviruses expressing the P2-HNF4α-BirA construct or a control empty vector. Cells were selected and amplified in the SILAC media as described above. To induce in vivo biotinylation of potential protein partners, cells were incubated 24 h in media containing 50 µM of biotin  before being collected by dissociation with trypsin and washed twice in D-PBS 1×. Nuclear extracts were prepared as described above and diluted in RIPA 1× buffer. To immunoprecipitate the biotinylated proteins, 50 µL of streptavidin coupled beads  was added to the nuclear extract and incubated 2 h at 4 °C. Beads were then washed three times with RIPA 1× and five times with 20 mM ammonium bicarbonate to remove unspecific interactions. After the last wash, the beads from the different conditions were combined and proteins directly converted to peptides using the on-Beads digestion technique described previously .P2-GFP HCT116 inducible cells were seeded in 48-well plates  at a density of 15,000 cells per well. The following day, cells were cultivated 48 h in the presence of 2.5 µg/mL of doxycycline  to induce the P2-GFP expression, or in the presence of an equivalent amount of water to serve as controls. Media were changed and cells were incubated 72 h in the presence of 3-fold dilution series of etoposide  ranging from 3.7 µM to 300 µM in the presence or not of doxycycline. Cell viability was determined with the alamarBlue Cell Viability Reagent following the manufacturer’s instructions (Invitrogen-Thermo Fisher Scientific). Fluorescence reading was achieved with the FlexStation 3 device  and cell viability was normalized to untreated control cells. Curve fitting was done with a four-parameter dose-response curve on the GraphPad Prism version 7.00  and two-way ANOVA was performed for statistical analysis.P2-GFP HCT116 inducible cells were seeded on a round glass coverslip (Thermo Fisher Scientific) in a 24-well plate to cover approximately 20% of the surface and induced 48 h with 2.5 µg/mL of doxycycline or not. Cells were fixed in 4% formaldehyde for 30 min and permeabilized 5 min in PBS Triton X-100 0.1%. Samples were blocked in PBS BSA 2% for 30 min and nuclei stained for 10 min in 0.3 µg/mL of DAPI. Coverslips were mounted on glass slides with Thermo Scientific Shandon Immu-Mount (Thermo Fisher Scientific) and cells were observed on a Zeiss Axiovert 200M fluorescent microscope . Homo sapiens proteome (88 354 entries) downloaded from the Uniprot database on the 07/16/2013. The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD008365. Trypsin digested peptides were loaded and separated onto a nanoHPLC system (Dionex Ultimate 3000). 10 µL of the sample (2 µg) was first loaded with a constant flow of 4 µL/min onto a trap column . Peptides were then eluted off towards an analytical column heated to 40 °C (PepMap C18 nano column 75 µm × 50 cm)) with a linear gradient of 5–35% of solvent B (90% acetonitrile with 0.1% formic acid) over a 4 h gradient at a constant flow (200 nL/min). Peptides were then analysed by an OrbiTrap QExactive mass spectrometer (Thermo Fischer Scientific) using an EasySpray source at a voltage of 2.0 kV. Acquisition of the full scan MS survey spectra (m/z 350–1600) in profile mode was performed in the Orbitrap at a resolution of 70,000 using 1,000,000 ions. Peptides selected for fragmentation by collision-induced dissociation were based on the ten highest intensities for the peptide ions from the MS survey scan. The collision energy was set at 35% and resolution for the MS/MS was set at 17,500 for 50,000 ions with maximum filling times of 250 ms for the full scans and 60 ms for the MS/MS scans. All unassigned charge states as well as singly, seven and eight charged species for the precursor ions were rejected, and a dynamic exclusion list was set to 500 entries with a retention time of 40 s (10 ppm mass window). To improve the mass accuracy of survey scans, the lock mass option was enabled. Data acquisition was done using the Xcalibur version 2.2 SP1.48 (Thermo Fischer Scientific). Identification and quantification of proteins identified by mass spectrometry was done using the MaxQuant software version 1.5.2.8  to score cm) withIn conclusion, our report identified interaction networks between HNF4α and multiple proteins characterized with a role in DNA repair. The precise mechanisms by which HNF4α acts during DNA repair biological processes must be complex and opens on a novel field of investigation for this central regulator involved in differentiation of endodermal-derived epithelial tissues as well as during the control of cell growth in tumorigenesis.


Swietenia macrophylla (Meliaceae), with its seed extract reported to display anti-hypoglycemic activities in diabetic rats. In the present study, three compounds isolated from the seeds of S. macrophylla were tested on a modified ELISA binding assay and showed to possess PPARγ ligand activity. They were corresponded to PPARγ-mediated cellular response, stimulated adipocyte differentiation but produced lower amount of fat droplets compared to a conventional anti-diabetic agent, rosiglitazone. The up-regulation of adipocytes was followed by increased adipocyte-related gene expressions such as adiponectin, adipsin, and PPARγ. The S. macrophylla compounds also promoted cellular glucose uptake via the translocation of GLUT4 glucose transporter.Numerous documented ethnopharmacological properties have been associated with  Swietenia macrophylla King (Meliaceae), commonly known as sky fruit or tunjuk langit by the locals, is a tropical timber tree native to tropical forests in America, Central America, South America and South-East Asia. Its fruit is consumed as a traditional remedy to promote blood circulation and general health. Over the years, numerous active constituents such as alkaloids, flavonoids and saponins have been extracted from S. macrophylla and their important ethnopharmacological properties were documented. These include anti-infective properties in pathogen, anticancer and anti-oxidation effects in cell-based and whole-organism model systems . F. F17]. Fde (6OA) . The pur3 solution. The 1H- and 13C-NMR spectra of the swietenine were recorded using a JEOL FT-NMR spectrometer (400 MHz). Gas chromatography-mass spectrometry (GC-MS) analysis was performed using an Agilent Technologies 6980 N gas chromatography system equipped with a 5979 Mass Selective Detector (70 eV direct inlet). An HP-5 ms (5% phenylethylsiloxane) capillary column (30.0 m × 25 mm × 25 µm) was used with helium as the carrier gas at flow rate of 1 mL/min. The column temperature was programmed initially at 150 °C, increased to 300 °C and held for 20 min at 5 °C per minute.The structure elucidation of the Sw was determined by spectral techniques. The compound was dissolved in a CDCl2CO3, 350 mM NaHCO3, pH 9.6). The plate was incubated for 2 h at room temperature. Nonspecific binding sites were blocked with 100 µL of blocking buffer  for 2 h at room temperature. The recombinant PPARγ cell lysate was prepared by using the freeze/thaw cell lysis method  after each incubation step.The ELISA binding assay used is based on the principle of binding between the ligand-dependent PPARγ and its coactivator. Briefly, 5 µg of purified recombinant p300, a PPARγ coactivator, was used to coat a 96-well microtitre plate in the presence of coating buffer  bovine serum and 10% (v/v) fetal bovine serum (FBS), respectively. The differentiation of adipocytes was initiated two days after the cells reached confluence by adding 0.5 mM isobutyl methylxanthine (IBMX), 1 µM dexamethasone and 1 µg/mL insulin to DMEM containing 10% (v/v) FBS. Two days later, the differentiation medium was replaced by DMEM containing 10% (v/v) FBS and 1 µg/mL insulin. These differentiated cells were stabilized in DMEM + 10% (v/v) FBS for another 2 days. C2C12 myocytes were induced to differentiate by substituting 10% (v/v) FBS with 10% (v/v) horse serum once the cells reached confluence. Fresh differentiation medium was replaced every two days until the myotube formation was complete.3T3-L1 and C2C12 cells were purchased from the American Type Culture Collection . The 3T3-L1 and C2C12 cells were maintained in DMEM medium supplemented with 10% , DMSO (negative control), TZD, and the three natural compounds isolated from S. macrophylla . The medium was changed every two days until >50% of differentiated cells were observed in the positive control treated wells. For visualization, lipid droplets were stained by Oil Red O (ORO). The trapped ORO between the lipid droplets was then dissolved in absolute isopropanol, and the absorbance of the dissolved ORO was semi-quantitatively measured by a SpectraMax M5 multi-detection microplate reader (Molecular Devices) at a wavelength of 510 nm.Adipocyte differentiation is one of the major cellular responses upon PPARγ activation. 3T3-L1 pre-adipocyte cells were seeded on 24-well plates at 5 × 10For the gene expression study, mature 3T3-L1 adipocytes were lysed 7 days after the induction of cellular differentiation. The total RNA samples were prepared according to the manufacturer's protocol . The mRNA expression of the target genes was measured by quantitative RT-PCR with an iCycler iQ5 PCR Thermal Cycler . The primers for the adipogenesis-related genes, adiponectin, adipsin, GLUT4 and PPARγ, were designed using Primer 3 software . Real-tiS. macrophylla compounds for 15, 30, 60 and 120 min in DMEM low glucose + 0.1% (w/v) BSA. The cells were then fixed with 4% (v/v) paraformaldehyde, washed with MAXwashTM washing medium  and blocked with MaxblockTM blocking medium (Active Motif). The cells were incubated with the GLUT4 antibody for 16 h at 4 °C and then with the fluorescent conjugated secondary antibody for 2 h at room temperature. 100 µL of FluoroshieldTM with DAPI  was added to each well and mounted with a cover slip for imaging.The differentiated C2C12 muscle cells cultured in 8-well chamber slides were treated with DMSO, insulin and S. macrophylla compounds for 60 min in DMEM + 0.1% (w/v) BSA. After washing three times with Krebs-Ringer Phosphate H (KRPH) buffer + 0.1% (w/v) BSA, the cells were incubated with 100 μM 2-NBDG for 15 min. After washing three times with KRPH buffer + 0.1% (w/v) BSA, the uptake of 2-NBDG was observed under a fluorescence microscope, and the fluorescence of 2-NBDG was measured with a microplate reader .C2C12 cells were grown and differentiated in 96-well culture plates. The differentiated C2C12 muscle cells were treated with DMSO, insulin, and p-values were considered significant at ∗p < 0.05, ∗∗p < 0.01.All the values were expressed as the means ±SD, and one-way ANOVA was used to determine the significance of the treatment effects at each time point. Differences among the treatment means were determined by Tukey’s test, and S. macrophylla were demonstrated to possess the properties of PPARγ ligands. The compounds exhibited significant binding activity to PPARγ, significantly increased levels of the mRNA expression of adipogenesis-related genes and induced adipocyte differentiation, though at a low level. The compounds induced the uptake of glucose by muscle cells via an increase in the translocation of GLUT4 to the plasma membrane. S. macrophylla bioactive compounds exhibited a good potential for anti-diabetic activity with a minimum side effect of weight gain.The three isolated bioactive compounds from


The authors apply telomeric chromatin analysis to identify factors that accumulate at telomeres during cellular transformation, promoting telomere replication and repair and counteracting oncogene-borne telomere replication stress. Telomeres play crucial roles during tumorigenesis, inducing cellular senescence upon telomere shortening and extensive chromosome instability during telomere crisis. However, it has not been investigated if and how cellular transformation and oncogenic stress alter telomeric chromatin composition and function. Here, we transform human fibroblasts by consecutive transduction with vectors expressing hTERT, the SV40 early region, and activated H-RasV12. Pairwise comparisons of the telomeric proteome during different stages of transformation reveal up-regulation of proteins involved in chromatin remodeling, DNA repair, and replication at chromosome ends. Depletion of several of these proteins induces telomere fragility, indicating their roles in replication of telomeric DNA. Depletion of SAMHD1, which has reported roles in DNA resection and homology-directed repair, leads to telomere breakage events in cells deprived of the shelterin component TRF1. Thus, our analysis identifies factors, which accumulate at telomeres during cellular transformation to promote telomere replication and repair, resisting oncogene-borne telomere replication stress. The identified proteins overlapped partially with proteins that were identified in previous telomeric proteomic screens (We compared the telomeric protein composition of the four HLF cell lines by QTIP  in a pai screens . These i screens . In addi screens  as 3xHA- screens  and S4. (A) List of all identified proteins. (B) List of telomeric proteins. (C) List of transformation-responsive telomeric proteins.Table S1 The four comparisons by QTIP  identifiThe telomeric long noncoding RNA TERRA has been implicated among others in the telomeric DDR and the regulation of telomerase . In addiTo test the hypothesis that during transformation, up-regulated proteins act against replicative stress at telomeres, we depleted candidate proteins  using siDetailed analysis of the metaphase spreads shown in Fig 5C. Displayed are percentages of chromosome ends  with normal and abnormal telomeric FISH signals and a number of scored telomeres and metaphases per condition. Fragile = smeary or multiple telomeric signals; Outside = telomeric signal positioned outside the DAPI-stained chromatid end; Apposition = sister telomere association/fusion; Fusion = non-sister telomere association/fusion.Table S2 SAMHD1 has been implicated in homology-directed repair of DNA double-strand breaks, recruiting CtIP to promote DNA end resection . In addiDetailed analysis of the metaphase spreads shown in Fig 6. Displayed are percentages of chromosome ends  with normal and abnormal telomeric FISH signals and a number of scored telomeres and metaphases per condition. Fragile = smeary or multiple telomeric signals; Outside = telomeric signal positioned outside the DAPI-stained chromatid end; Apposition = sister telomere association/fusion; Fusion = non-sister telomere association/fusion.Table S3 We then tested if SAMHD1 depletion also triggers outsider telomeres in cells in which telomere fragility was induced by treatment with the DNA polymerase α and δ inhibitor aphidicolin . Chromatin-enriched pellets were resuspended at 25 × 106 cells/ml in LB3 buffer  and sonicated for 12 min at 4°C using the Focused-ultrasonicator . Immunoprecipitation was performed using beads covalently coupled with nonspecific IgGs  or beads coupled to affinity-purified anti-TRF1 (rabbit #714) and anti-TRF2 antibodies (rabbit #40) according to the previously published protocol . Further statistical analyses and graphical displays were performed in Perseus software version 1.5.3.2 (http://www.coxdocs.org/), using mean values of the forward and reverse QTIP replicates. The expression values and ratios were log2-transformed and normalized by subtracting the median. Proteins enriched in TRF IP over control IgG IP  were calculated separately for each QTIP as statistically significant outliers using a Significance A left-sided test (P < 0.05). Only proteins with significant TRF/IgG ratio in at least two of four QTIPs were considered “telomeric.” Upon manual inspection, proteins HRG and SERPINC1 were removed from the list of telomeric proteins because they were light labeled in both label-swap experiments, indicating they were external contaminants. Significant SILAC ratios were determined for each QTIP  using a Significance B two-sided test . Proteins with a significant SILAC ratio in at least one of the four QTIPs were considered “transformation responsive.” To create heatmaps, hierarchical clustering was performed in Perseus based on average Euclidean distance of the log2 mean SILAC ratios. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  by calcium phosphate precipitation twice with a 36-h interval and harvested 80–100 h after the first transfection. siRNA oligonucleotides were purchased from Qiagen: SAMHD1 , DCLRE1B , TMPO , NPAT , PARP9 , PNUTS , and SMCHD1 (SI00454664 and SI00454678). siGFP 5′-GCAGCACGACTTCTTCAAGTT-3′ was synthesized at Microsynth. For transient overexpression of 3xHA-tagged proteins, HEK293T cells in a 15-cm tissue culture dish were transfected with 30 μg of plasmid DNA using calcium phosphate precipitation. Cells were harvested for ChIP analysis 42–48 h post-transfection. HeLa cells were transfected with pSuper-Puro and pSuper-Blast constructs using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. 1 μg/ml puromycin and 5 μg/ml blasticidin were added to the medium 1 d after the transfection, and selection was maintained for 5 d.6 cells/ml), and sonicated for 30 min at 4°C using a Focused-ultrasonicator as described in the QTIP protocol. The sonicated extracts were centrifuged at 4°C for 15 min at 20,000 g and the supernatant was mixed with four volumes of ChIP dilution buffer and precleared for 1 h at 4°C with Sepharose 6B (Sigma-Aldrich) pre-blocked with yeast tRNA (0.5 mg/ml of beads). The precleared lysate corresponding to 2 × 106 cells was supplemented with 40 µl of yeast tRNA-blocked Protein G 50% bead slurry  and 3 μg of anti-HA antibody . After an overnight incubation at 4°C, the beads were washed as for QTIP. The DNA from input and IP samples was isolated and analyzed by dot blot hybridization as described previously .RNA extraction was performed using RNeasy Mini kit (Qiagen) according to manufacturer’s protocol. Northern blot TERRA analysis was performed as described previously . In brie8 to 3 × 10−1. The concentration of each plasmid was determined using Qubit 4 Fluorometer (Invitrogen). We calculated the copy number concentration using the following formula:To determine the copy number of TERRA in the qPCR reactions, we constructed a standard curve for each TERRA based on a recombinant plasmid containing the desired subtelomere sequence. The subtelomeres were amplified from HeLa genomic DNA using Phusion Green High-Fidelity DNA polymerase and the primers listed in Table S4. The PCR products were isolated from the gel, purified using the Qiagen gel extraction kit, and cloned in the pCR4 BLUNT-TOPO vector using the Zero Blunt TOPO PCR cloning kit for sequencing. All the plasmids were sequenced to confirm the subtelomere sequence. To construct the standard curves for each TERRA, we made a 10-fold serial dilution of each plasmid ranging from 3 × 10List of primers for RT–qPCR of TERRA.Table S4 T values were plotted against the logarithm of the plasmid copy number and the standard curve was generated by a linear regression of the points. The PCR amplification efficiency was calculated as a percentage from the slope of the curve using the following formula:Each standard curve was performed in duplicate. The C−ΔΔCt method. The number of TERRA copies in the qPCR or the cDNA template was calculated based on the standard curve.RNA was isolated using RNeasy Mini kit (Qiagen) according to the manufacturer’s protocol. The RNA was reversed-transcribed using the Invitrogen SuperScript III Reverse Transcriptase as previously described . The cDN15 primers (Promega). The qPCR was performed as previously described (3 μg of RNA was reverse-transcribed using SuperScript III RT (Invitrogen) with 150 ng random primers (Promega) and 500 ng oligo (dT)escribed  using prList of primers for RT–qPCR to verify candidate depletion.Table S5 3 PNA probe. The spreads were denatured at 80°C for 3 min and hybridized at RT for 3 h. The slides were washed twice for 15 min with 10 mM Tris–HCl (pH 7.4)/70% formamide, and three times for 5 min with 0.1 M Tris–HCl (pH 7.4)/0.15 M NaCl/0.08% Tween-20 with DAPI in the second wash. The slides were dehydrated in an ethanol series and mounted in Vectashield embedding medium. Images were acquired using a Zeiss Axioplan II microscope.Cells were treated with 0.05 μg/ml demecolcine for 2 h, harvested, and incubated in 0.056 M KCl at 37°C for 7 min. Swollen cells were fixed in cold methanol/acetic acid (3:1) overnight and spread on glass slides. After ageing overnight, the slides were rehydrated in 1× PBS, fixed in 4% formaldehyde, and dehydrated in an ethanol series. The slides were allowed to air-dry before applying the hybridization solution  containing Cy3-OO-(CCCTAA)3 PNA probe as described above. Images were captured using a Zeiss LSM 700 confocal microscope.Cells were grown on coverslips, fixed in 4% formaldehyde, and permeabilized for 5 min in 0.1% Triton X-100/0.02% SDS/1× PBS, and then pre-blocked for 10 min in 2% BSA/1× PBS and blocked for 45 min in 10% goat serum/2% BSA/1× PBS. Cells were then incubated with 0.5 μg/ml α-53BP1 antibody  for 1 h, washed three times for 4 min in 2% BSA/1× PBS, and incubated with Alexa Fluor 633–conjugated secondary antibody  for 30 min. After three washes in PBS, stained cells were fixed in 4% formaldehyde for 5 min. Both antibodies were diluted in 10% goat serum/2% BSA/1× PBS, and all steps of the IF protocol were performed at RT. For telomeric FISH, the stained and fixed coverslips were dehydrated in an ethanol series, air-dried, and hybridized with the Cy3-OO-(CCCTAA)32P]-dCTP–labeled telomeric probe at 50°C as described (Genomic DNA was isolated using the Wizard Genomic DNA Purification kit (Promega) and subjected to restriction digestion with RsaI and HinfI, in the presence of RNase A, at 37°C overnight. Digested DNA was extracted using phenol–chloroform–isoamyl alcohol, followed by isopropanol precipitation. 1 μg of digested DNA was separated on 1% agarose gel by pulsed-field gel electrophoresis using a Bio-Rad CHEF DR-II apparatus or on 0.8% agarose gel by constant field gel electrophoresis. The gels were dried and hybridized with [αescribed . RadioacImmunoblots were incubated separately with the following primary antibodies: anti-ATM pS1981 , anti-Chk2 pT68 , anti-p53 , anti-p21 , anti-Actin , anti-TRF2 , and anti-γH2AX , followed by a corresponding horseradish peroxidase–conjugated secondary antibody.5 cells was resuspended in 250 µl of PBS containing 0.2 μg/ml RNAse A and incubated for 15 min at 37°C. The DNA was stained by adding 250 µl of 80 μg/ml of propidium iodide, followed by 10-min incubation at 4°C in the dark. 250 µl of PBS was added to each sample before FACS analysis on Accuri C6 (BD Biosciences). The percentage of cells in each phase of the cell cycle was determined using the Watson Pragmatic computational model in FlowJo software (TreeStar).Following fixation in 70% ethanol, the pellet from 5 × 106 cells were harvested for each condition and lysed for 5 min in cold RLN buffer  supplemented with 1 mM DTT and 100 U/ml RNasin Plus (Promega). After centrifugation , the nuclei-enriched pellet was resuspended in RLT buffer  supplemented with βmercaptoethanol at 10 µl/ml and homogenized by passing through an insulin needle. Nucleic acids were thereafter isolated by a phenol:chloroform:isoamylalcohol  separation, followed by isopropanol precipitation. The nucleic acids were dissolved in H2O  and sonicated using a Focused-ultrasonicator (Covaris E220) to obtain DNA fragments below 500 bp. 150 µl of sonicated nucleic acids were treated for 90 min at 37°C with either 10 µl of RNase H  or H2O. The reaction was stopped by addition of 2 µl of 0.5 M EDTA. The samples were mixed with 1,738 µl of buffer 1  and precleared for 1 h at 4°C with 80 µl of 50% Protein G bead slurry  that had been pre-blocked for 1 h at 4°C with yeast tRNA (1 mg tRNA per 1 ml of 50% bead slurry). The IP reactions were set up containing 800 µl of precleared extract, 40 µl of tRNA-blocked Protein G beads, and 1 μg of S9.6 antibody  or mouse IgG . The reactions were incubated for 90 min at 4°C on a rotating wheel and then washed consecutively for 5 min each time with 1 ml of buffer 2 , buffer 3 , buffer 4 , and 1× TE buffer . The input and precipitated DNA was analyzed by dot blot hybridization.60–80 × 103 primer containing thiophosphate linkages between the three 3′ terminal nucleotides. The mix was denatured at 96°C for 5 min and cooled down to 25°C for 2 h. DNA was ethanol-precipitated and resuspended in 10 μl of H2O for the T-circle reaction . Primer extension was carried out at 30°C for 12 h. The ϕ29 DNA polymerase was inactivated by incubation at 65°C for 20 min. The extension products were separated by agarose gel electrophoresis (0.6% agarose at 2 V/cm for 16 h). The gel was then dried at 50°C for 2 h, denatured and hybridized with a specific telomeric probe. U2OS genomic DNA of the ALT cell line was used as a positive control.T-circle assay was modified from the work of https://doi.org/10.26508/lsa.201800121.Supplementary Information is available at


Background: The ERK MAPK pathway plays a pivotal role in regulating numerous cellular processes during normal development and in the adult but is often deregulated in disease scenarios. One of its key nuclear targets is the transcription factor ELK1, which has been shown to play an important role in controlling gene expression in human embryonic stem cells (hESCs). ELK1 is known to act as a transcriptional activator in response to ERK pathway activation but repressive roles have also been uncovered, including a putative interaction with the PRC2 complex.Methods: Here we probe the activity of ELK1 in hESCs by using a combination of gene expression analysis in hESCs and during differentiation following ELK1 depletion and also analysis of chromatin occupancy of transcriptional regulators and histone mark deposition that accompany changes in gene expression.Results: We find that ELK1 can exert its canonical activating activity downstream from the ERK pathway but also possesses additional repressive activities. Despite its co-binding to PRC2 occupied regions, we could not detect any ELK1-mediated repression at these regions. Instead, we find that ELK1 has a repressive role at a subset of co-occupied SRF binding regions.Conclusions: ELK1 should therefore be viewed as a dichotomous transcriptional regulator that can act through SRF to generate both activating and repressing properties at different genomic loci. In vitro studies on human embryonic stem cells are an important step in understanding the molecular basis to human development. Cultured human embryonic stem cells (hESCs) require FGF2-mediated signalling through the ERK pathway to maintain their pluripotent state , although the majority of ELK1 binding peaks showed no overlap  Venn diagram showing the intersection of binding regions from genome-wide chromatin immunoprecipitation-sequencing datasets for ELK1, SUZ12 and SRF in H1-hESCs. (B andC) Transcription factor and histone modification tag density profiles in H1-hESCs in a region 2,500 bp to either side of the centre of the ELK1 binding regions. ELK1 binding regions are portioned according to (A) overlapping with SUZ12 binding regions (+SUZ12) or (B) not overlapping with SUZ12 binding regions (-SUZ12). (D andE) diagrammatic illustration of the ELK1 binding regions associated with co-binding of the repressive PRC2 complex (D) or associated with its binding partner SRF and active regions of chromatin (E). (F andG) Biological function gene ontology terms of genes associated with ELK1 binding loci either overlapping with SUZ12 binding loci (D) or not overlapping with SUZ12 binding loci (E). to active during differentiation. We chose to use retinoic acid (RA), a potent initiator of hESC differentiation. Using the nearest TSS-association model in HOMER, we identified a set of ELK1+SUZ12-bound genes whose expression increased upon 48–96 hours of treatment with RA and focussed on six of these that showed robust induction .ELK1 exditions . To undeditions  and examditions . Howeverditions , nor didditions . Thus, aditions  and ChIPditions  data are-209). Notably, there are very few regions co-bound by ELK1, SRF and SUZ12, indicating a clear distinction between regions bound by ELK1 and either SRF or SUZ12. We decided to focus on the role of ELK1 in differentiation to mesoderm as SRF has previously been implicated in this developmental process  exhibited increased expression following differentiation to mesoderm, suggesting a potential repressive function in this context  also show SRF co-occupancy  on regions which were bound by both ELK1 and SRF. Binding of both factors was specifically detectable on the known target genesndEGR2 . GeneralndEGR2 . This pandEGR2  and 18/2ndEGR2 . This isndEGR2 . This pandEGR2  and 16 ondEGR2 . These rndEGR2 .EGR1,EGR2 andEGR3 in these cells. As these genes are inducibly activated by ERK pathway signalling, we activated the ERK pathway by culturing mesoderm cells (derived from H1-hESCs) in DMEM/F12 media followed by serum starvation for 24 hrs and then switched the media to MIM for 15 mins. This treatment caused increased levels of active, phosphorylated ERK  were routinely cultured in mTeSR1™ (StemCell Technologies). Plates were coated with Matrigel (Corning) at 37°C for 1 hour before passage. To passage the cells, the cells were coated with a thin layer of ReLeSR™ (StemCell Technologies) and incubated at 37°C for 5 minutes.For shELK1 and RA treatment, conditioned H1 media was used, containing DMEM/F12 (Invitrogen), 20% (v/v) knockout serum replacement (Thermo Fisher Scientific), 1 mM L-glutamine (Gibco), 1% (v/v) nonessential amino acids (Gibco), 0.1 mM 2-mercaptoethanol (Gibco), and 4 ng/ml basic fibroblast growth factor (Invitrogen). This media was conditioned with CF1 mouse fibroblasts  for 24 hr prior to adding to the H1-hESCs. Media was then vacuum filtered (0.22 µM), and an additional 8 ng/ml of basic fibroblast growth factor (Invitrogen) was supplemented to conditioned medium before usage. To passage the cells, the cells were coated with a thin coat of dispase (StemCell Technologies) and incubated at 37°C for 5 minutes.5 per cm2 in mTeSR1™ supplemented with 10 µM Y-27632. After 24 h, cells were grown in STEMdiff™ mesoderm Induction medium (StemCell Technologies) for an additional 3 days.For differentiating hESCs to mesoderm, cells were first treated with 10 µM Y-27632 (ROCK inhibitor) for 1 hour before passage and dissociated with TrypLE™ Express (StemCell Technologies). Cells were initially seeded at a density of 5×10To activate the ERK signalling pathway, H1-hESCs were first allowed to differentiate into mesoderm cells, maintained in DMEM/F12 media lacking serum for 24 h and then switched the media to MIM for 15 mins for analysing ERK activation and 60 mins for studying gene expression. DMEM/F12 media was used as a control for MIM stimulation.5 per cm2 in mTeSR1™ supplemented with 10 µM Y-27632. After 24 h, cells were grown in in mTeSR1™ supplemented with 5 µM RA (Sigma) for up to 96 h. DMSO was used at a final concentration of 1:10,000 as a control for RA treatment.For retinoic acid (RA) treatment, cells were dissociated with TrypLE™ Express and seeded at a density of 5×10For cytochalasin D stimulation,  H1-hESCs were first allowed to differentiate into mesoderm cells, maintained in DMEM/F12 media lacking serum for 24 h and then switched to DMEM/F12 containing 2 μM  cytochalasin D for 60 minutes to study gene expression. DMEM/F12 media containing DMSO at a final concentration of 1:10,000 was used as a control for cytochalasin D stimulation.5 cells were treated with 7.5 µl of TransIT®-LT1 (Mirus) plus 2.5 µg of shRNA plasmid prepared in 250 µl of OptiMEM. shELK1 plasmid was a pSuper-derived plasmid containing the shRNA hairpin for ELK1: 5’-GCCAGAAGTTCGTCTACAA-3’  and 2.5 µl of siRNA (20 µM stock concentration), prepared in 150 µl Opti-MEM (Gibco). A non-targeting siRNA (siNT) was used as a control for siELK1 and siSRF treaments.For siRNA transfection cells were prepared as above and after dissociation seeded at a concentration of 5×106 cells were seeded per immunoprecipitation (IP)). The crosslinking reaction was then quenched with 0.125 M glycine for 5 minutes. Cells were washed with ice cold 1x PBS. Next, 3×106 cells were harvested in FA cell lysis buffer  Triton-X100, 10 mM EDTA, 0.1M NaCl) rotated for 10 minutes at 4°C, the nuclei pelleted at 13.1 krpm at 4°C for 5 minutes and the supernatant discarded. Cells were resuspended in FA Cell Lysis Buffer, rotated for 10 minutes at 4°C, pelleted at 13.1 krpm at 4°C for 5 minutes and the supernatant discarded. Nuclei were then resuspended in 1% SDS solution  Triton-X100, 0.1% (v/v) Na-DOC, 1% (w/v) SDS), rotated for 10 minutes at 4°C and the chromatin pelleted at 13.1 krpm at 4°C for 5 minutes and the supernatant discarded. Chromatin was then suspended in 0.1% SDS solution  Triton-X100, 0.1% (v/v) Na-DOC, 0.1% (w/v) SDS), rotated for 10 minutes at 4°C, pelleted at 13.1 krpm at 4°C for 5 minutes and the supernatant discarded. Chromatin was then resuspended in 0.1% SDS solution and sonicated to produce chromatin fragments of 100–500 bp.Cells were incubated at room temperature with 1% (v/v) formaldehyde (Sigma), for 10 minutes  and 1.25 µg of antibodies were conjugated at room temperature for 2 hours, after which, conjugated beads were washed with 0.1% SDS buffer. The lysate was then rotated with the conjugated beads overnight at 4°C. The next day, beads were washed sequentially with 0.1% SDS solution high salt wash  Triton-X100, 0.1% (v/v) Na-DOC, 0.1% (w/v) SDS), NP40/LiCl wash  NP-40, 0.1% (w/v) Na-DOC)and TE . The beads were then resuspended in ChIP elution buffer  SDS) and shaken at 69°C at 1000 rpm for 1 hour. This supernatant was then transferred to a new tube, treated with 1:50 Proteinase K (20 mg/ml) (Roche) and shaken at 55°C at 600 rpm for 1 hour. For siELK1 and MIM treatment, DNA was then further purified using the QIAquick PCR purification kit (Qiagen). For shELK1 and RA treatment, the eluted DNA was mixed with an equal volume of phenol-chloroform (Thermo Fisher Scientific). The aqueous layer was isolated following centrifugation, for 10 minutes at 13.1 krpm at 4°C, and mixed 1:1 with isopropanol and frozen at -80°C for 30 minutes. The solution was then spun at 13.1 krpm at 4°C for 20 minutes and the supernatant discarded. The pellet was then washed with 70% ethanol and spun two more times. It was then air-dried for 24 h and 100 µl of water was added.For 3×10Extended data, Supplementary Table S1 (A PCR reaction was then run with the following settings: 50°C for 30 min (only for RT-PCR), followed by 95°C for 20 min then  for 40 cycles, melt curve 72-95°C. ChIP-qPCR samples inable S1 . Extended data, Supplementary Table S1  using the manufacturer’s protocol. For RA and shELK1 experiments, cells were collected into 350 µl of RNAzol (Sigma) homogenised with a Gilson pipette, and spun at 13.1 krpm at 4°C for 20 minutes The aqueous layer was then mixed 1:1 with isopropanol and RNA precipitated and dried as described for ChIP-isolated DNA above. cDNA was then prepared using SuperScript2 (Thermo Fisher Scientific) according to the manufacturer’s instructions (PCR was then carried out using Power SybrGreen (Thermo Fisher Scientific) with an annealing temperature of 55°C, according to the manufacturer’s instructions. Data was collected with the ViiA 7 (Thermo Fisher Scientific) and analysed with Viia7 V1.2 software (Thermo Fisher Scientific). The PCR primers are shown inable S1 .Extended data, Supplementary Table S2  and measured against BSA standards ranging from 0.2 mg/ml to 2 mg/ml. Approximately 20 µg of protein was used for each well. 1x SDS loading buffer was added to the lysate, which was then boiled at 99°C for 10 minutes. Proteins were resolved on the 12% gel in 1x SDS running buffer, and transferred to a nitrocellulose membrane using transfer buffer. Finally, cells were incubated with primary and secondary antibodies (able S2 ), diluteExtended data, Supplementary Table S3 (Bowtie2 (v2.2.9) with default settings (bedtools (v2.21), reads ettings . Bowtie2 (v2.21), using tHOMER (v4.8.3) .Data are available under the terms of the The authors have satisfactorily responded to my comments. While they have not ruled out a role for Elk1 repression through blocking MRTF-A/B binding to SRF, they have qualified their conclusions.   In general this is an interesting study showing Elk1 repression of target genes co-bound by SRF. The mechanism of Elk1 repression remains to be determined.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The authors have addressed appropriately all my concerns.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. 1) in which ERK genome-wide chromatin interactions in human embryonic stem cells was characterized. In the present paper, the authors observed a significant overlap in ELK-1 and SUZ12 (PRC2 complex) binding regions but they don’t find any evidence that ELK-1 does have a role in mediating transcriptional repression through these regions. Interestingly they find that ELK-1 has a repressive role during mesoderm differentiation that appears not to be exerted through competition with MRTF co-activators of RhoA-mediated gene expression. The paper represents an interesting follow up of the earlier work and is written in a clear and logical fashion. The purpose of the study is well defined and technically sound. However some data and aspects of the manuscript are worth clarifying.In this paper, the authors evaluate the dual activating and repressive role of the ETS transcription factor ELK-1 in human embryonic stem cells. The paper builds upon a previous study  do not have this ELK-1 binding motif, at least for a 500bp window1. Is this also the case with your 2500bp window? If true, do the authors have any explanation of how ELK-1 is recruited?Figure 2A. The RT-qPCR experiments after retinoic acid (RA) treatment appear somewhat highly variable especially for PAX7 and POUF3F2. Do the authors identify more than the six ELK-1+SUZ12 bound genes whose expression increased upon RA treatment? If yes, it should be worth to include them. Is the expression of ELK-1 affected by RA?Figure 3. The role of ELK-1 during mesoderm expression should be discussed in more detail. The authors nicely show that ELK-1 depletion caused an increase in the expression of FOXC1 and HAND2  but in the meantime the Western-blot  suggests that ELK-1 protein expression is induced during mesoderm differentiation. Is this possible that these genes are ELK-1/PRC2- mediated repression dependent?Figure 5. FOXC1  is included in the heatmaps of chromatin Ip but not HAND2. If the ChiP-qPCR has been done for HAND2, it could be nice to add it.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The authors nicely evaluate the role of Elk1 in regulating gene expression in human embryonic stem cells. Elk1 co-binds to DNA with SRF, but can also bind DNA without SRF. They don’t find evidence that the sites bound without SRF are functional here. Interestingly, they find that Elk1 can function as an activator or a repressor depending upon the exact gene binding site they examine. One conclusion that I feel is less well proven is that Elk1 repression is independent of its blocking another SRF co-factor MRTF-A and -B. Their evidence is that activation of MRTF-A/B by cytochalasin D is not stimulated after Elk1 knockdown. However, the activation of specific genes in these cells could be due to basal MRTF-A/B activation (or is the related myocardin gene expressed in these cells?). If so, perhaps there is something about these cells that limits cytochalasin activation of MRTF-A/B at these genes. A more thorough way to test this question is to knockdown the members of the MRTF family along with Elk1 knockdown to see whether the derepression is affected.In Figure 6, different cell treatments are used in 6B and 6D, E. The differences are not entirely clear. In 6B, there is a brief treatment of MIM media. What is done in the other figures? This is important as in 6B, Elk1 activates expression and in 6E it represses.It’s concluded for Fig. 6, on page 6, that Elk1 represses specific genes. This implies that it is doing so directly by binding to them. What rules out an indirect effect? This should at least be discussed.In Figure 2, the authors examine retinoic acid induction of the cells and conclude that Elk1 binding to PRC2 is not important. Later in the paper they use media to induce the cells to differentiate to mesodermal cells. Is it possible that Elk1/PRC2 target genes are important for this type of induction of these cells?1 and found a redundancy for immediate early activation of some genes by Elk1 and MRTF. Are the embryonic stem cells different than other cell types for Elk1 activity and does the growth state of the cells affect Elk1 activity?We previously found that Elk1 could act as a repressor of MRTF target genes in NIH3T3 cells particularly when grown continuously Some more minor points:I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.


The cytotoxicity of astrocytes was nimodipine-dependently reduced during osmotic (p ≤ 0.01), oxidative (p ≤ 0.001) and heat stress (not significant). Moreover, a decreased caspase activity as well as an increased proteinkinase B (AKT) and cyclic adenosine monophosphate response element-binding protein (CREB) phosphorylation could be observed after the nimodipine treatment under different stress conditions. These results demonstrate a cell type-independent neuroprotective effect of the prophylactic nimodipine treatment, which is associated with the prevention of stress-dependent apoptosis through the activation of CREB and AKT signaling pathways and the reduction of caspase 3 activity.Clinical and experimental data assumed a neuroprotective effect of the calcium channel blocker nimodipine. However, it has not been proven which neuronal or glial cell types are affected by nimodipine and which mechanisms underlie these neuroprotective effects. Therefore, the aim of this study was to investigate the influence of nimodipine treatment on the in vitro neurotoxicity of different cell types in various stress models and to identify the associated molecular mechanisms. Therefore, cell lines from Schwann cells, neuronal cells and astrocytes were pretreated for 24 h with nimodipine and incubated under stress conditions such as osmotic, oxidative and heat stress. The cytotoxicity was measured via the lactate dehydrogenase (LDH) activity of cell culture supernatant. As a result, the nimodipine treatment led to a statistically significantly reduced cytotoxicity in Schwann cells and neurons during osmotic ( The calcium antagonist nimodipine binds to the α1-subunit of calcium channels and regulates the calcium influx. Nimodipine treatment is used to reduce vasospasm in relation to the management of aneurysmal subarachnoid hemorrhages (aSAHs) as well as having shown a positive effect on the outcome and the prevention of accompanied delayed ischemic neurological deficits (DIND) . Furtherp ≤ 0.001; p ≤ 0.001, p ≤ 0.01; p ≤ 0.01) in comparison to the vehicle cells. The treatment with 1 µM nimodipine showed no significant alteration of the Schwann cell cytotoxicity under heat stress.The cytotoxicity was measured via lactate dehydrogenase (LDH) activity in cell culture supernatants after the treatment of cells with 150 mM NaCl (osmotic stress), 2% EtOH (oxidative stress) and incubation for 6 h at 42 °C (heat stress). The Schwann cells showed a nimodipine-dependent significant reduction of cytotoxicity up to 19% at osmotic stress  and 21% . The treatment with 1 µM nimodipine was not significantly altered, when compared to the vehicle. During oxidative stress, the cytotoxicity was reduced from 45% to 30% (p ≤ 0.001) but only slightly under heat stress (p ≤ 0.05) compared to the control cells (vehicle). The treatment with 1 µM nimodipine did not lead to a significant difference of neuronal cell toxicity under oxidative and heat-related stress.The results of the neuron LDH release analysis demonstrated a decreased cytotoxicity of immortalized neurons at osmotic stress from 31% to 26% (p ≤ 0.01) and 14% (20 µM nimodipine p ≤ 0.05). After the incubation of the cells with 2% ethanol (oxidative stress), the results showed a decreased cell death of up to 25% (p ≤ 0.001). Under heat stress, a reduction of the cytotoxicity of up to 17% , to approximately 60% during oxidative stress (10 µM and 20 µM nimodipine p ≤ 0.001) and to approximately 90% during heat stress (10 µM and 20 µM nimodipine p ≤ 0.001) for the Schwann cells compared to the vehicle cells. The neuronal cells showed a caspase activity reduction by 36% during osmotic stress (10 µM and 20 µM nimodipine p ≤ 0.001), to 82% and 70% during oxidative stress (10 µM and 20 µM nimodipine p ≤ 0.001) as well as to approximately 90% (p ≤ 0.001) during heat stress compared to the vehicle control. For the astrocytes, the caspase 3/7 activity was reduced to ~60% during osmotic (10 µM and 20 µM nimodipine p ≤ 0.001), to 71% during oxidative (20 µM nimodipine p ≤ 0.01) and to 75% during heat stress (10 µM nimodipine p ≤ 0.001) in comparison to the vehicle. The 20 µM nimodipine treatment led to an increased caspase 3/7 activity under heat stress in the astrocyte cell line .The influence of the nimodipine treatment on the caspase 3 and 7 activity of stressed cells was detected via Caspase-Glo 3/7 Assay (Promega). The analysis showed an alteration of caspase 3 and 7 activity through the nimodipine treatment under osmotic, oxidative and heat stress in Schwann cells A, neuronThe immunoblot analysis of the nimodipine-treated SW10 cells showed an increased AKT phosphorylation at serin residue 473 and CREB phosphorylation at serin residue 133 , which wNimodipine is a well-tolerated drug with a good safety profile. Because of its lipophilic properties, nimodipine passes the blood-brain barrier in a better way than other calcium antagonists . For cerVery Early Nimodipine Use in Stroke (VENUS) trial showed no beneficial effect of nimodipine treatment when nimodipine was applied 6 h after an acute stroke [In the last years, some clinical trials have shown a beneficial effect of nimodipine treatment for the preservation and regeneration of nerve function after resections of vestibular schwannoma and maxillofacial surgery ,5,6. Fure stroke . But dife stroke ,22. Matte stroke . They dee stroke . Furthere stroke . These de stroke ,12,26. Ee stroke . The proe stroke ,29. Osmoe stroke ,31. In 2In previous studies, nimodipine concentrations between 1–20 µM were appropriate for achieving a neuroprotective effect in cell culture ,12, whicCaspase 3 and 7 are effector caspases, irreversibly inducing the apoptosis and doing so in a direct manner via the degradation of the cell membrane and of the cytoskeleton actin filaments ,38,39,40The analysis of the cell signaling pathways of Schwann cells has shown a nimodipine-dependent increased AKT phosphorylation at serin 473 without stress and osmotic, oxidative or heat stress . The AKTAs a voltage-dependent L-type calcium channel blocker, nimodipine regulates the calcium influx in the cells ,57, whic2 at 37 °C. The neuronal cells were cultured in DMEM/F12  containing the same supplements at 5% CO2 and 33 °C. The cell lines were routinely analyzed for mycoplasma contamination. All experiments were carried out in three independent biological replicates.The murine cell lines C8-D1A , RN33B  and SW10  were obtained from the American Type Culture Collection . Astrocytes and Schwann cells were cultured in Dulbecco’s Modified Eagle’s Medium , supplemented with 10% fetal calf serum , 2 mM glutamine , 100 U/mL penicillin and 100 μg/mL streptomycin (ThermoFisher Scientific) with 5% CO4 SW10 and 5 × 104 C8-D1A or RN33B cells were seeded in 24-well plates and treated 24 h prior to the stress application with 1 µM, 10 µM and 20 µM nimodipine [1 × 10modipine  , as previously described in detail . For the24 h after the stress induction , the cel4 cells were seeded in white 96-well cell culture plates  in triplicates and were pretreated for 24 h with 1, 10 and 20 μM nimodipine or ethanol alone (solvent control). 24 h after the stress induction with 150 mM NaCl (osmotic stress), 2% EtOH (oxidative stress) or incubated 6 h at 42 °C (heat stress), the cells were incubated for 30 min with 100 μL Caspase-Glo 3/7 reagent in the dark, followed by a measurement of the luminescence signals . A cell-free culture media served as the background control. Signals of untreated stressed cells were set to 100%. The results showed the means ± SD of three technical replicates.The activity of caspase 3 and 7 was measured using a Caspase-Glo 3/7 assay , according to the manufacturer’s instructions. 1 × 10p values were ≤0.05 . The data were expressed as the mean ± S.D.The statistical analysis was performed with a one-way ANOVA followed by a Tukey’s post hoc test . The significance was accepted if the Nimodipine pretreatment leads to cell type-independent neuroprotection through the decreased activation of caspase 3 and 7 and the increased activation of CREB and AKT signaling . This ce


The crystal structures of sodium dirubidium citrate and sodium dirubidium citrate dihydrate have been solved and refined using laboratory X-ray powder diffraction data, and optimized using density functional techniques. Both structures contain Na chains and Rb layers, which link to form different three-dimensional frameworks. 2(C6H5O7)]n} and sodium dirubidium citrate dihydrate {poly[diaqua(μ-citrato)dirubidium(I)sodium(I)], [NaRb2(C6H5O7)(H2O)2]n} have been solved and refined using laboratory X-ray powder diffraction data, and optimized using density functional techniques. Both structures contain Na chains and Rb layers, which link to form different three-dimensional frameworks. In each structure, the citrate triply chelates to the Na+ cation. Each citrate also chelates to Rb+ cations. In the dihydrate structure, the water molecules are bonded to the Rb+ cations; the Na+ cation is coordinated only to citrate O atoms. Both structures contain an intramolecular O—H⋯O hydrogen bond between the hydroxy group and one of the terminal carboxylate groups. In the structure of the dihydrate, each hydrogen atom of the water molecules participates in a hydrogen bond to an ionized carboxylate group.The crystal structures of sodium dirubidium citrate {poly[μ-citrato-dirubidium(I)sodium(I)], [NaRb A systematic study of the crystal structures of Group 1  citrate salts has been reported in Rammohan & Kaduk 2018. The stu2C6H5O7, the root-mean-square deviation of the non-hydrogen atoms in the refined and optimized structures is 0.095 Å 2, the agreement of the refined and optimized structures is poorer , which is one of the two low-energy conformations of an isolated citrate , with a bond-valence sum of 1.19. The Rb20 and Rb21 cations are eight- and nine-coordinate, respectively. The coordination polyhedra are irregular, and the bond-valence sums are 0.94 and 1.03. The Mulliken overlap populations in both structures indicate that the Rb—O bonds are ionic, but that the Na—O bonds have some covalent character.In the dihydrate, the citrate anion similarly triply chelates to Na19 through the terminal carboxylate O12, the central carboxylate O15, and the hydroxy group O17 . Each terminal carboxylate group chelates to a different Rb21 cation. Most of the oxygen atoms bridge multiple metal atoms, but O13 and O14 bind only to Rb cations, and O17 binds only to the Na2C6H5O7 2 2 Fig. 6, the NaO2C6H5O7, the only traditional hydrogen bond is an intramolecular O17—H18⋯O11 one between the hydroxyl group and one of the terminal carboxylate groups 2, each water molecule hydrogen atom acts as a donor in an O—H⋯O hydrogen bond to a carboxylate oxygen 2 yielded 21 hits, but when including the chemistry of C, H, Na, O, and Rb only it yielded no hits.Details of the comprehensive literature search for citrate structures are presented in Rammohan & Kaduk 2018. A reduc2C6H5O7(H2O)2 was prepared by adding stoichiometric quantities of Na2CO3 and Rb2CO3 to a solution of 10 mmol H3C6H5O7 in 10 ml of water. After the fizzing subsided, the clear solution was dried overnight at 348 K to yield a glass. This glass was heated at 450 K for 30 min to yield a pale-yellow solid. This solid was equilibrated in air at ambient conditions for 3 h. The anhydrous salt was prepared by heating the dihydrate at 450 K for 30 min.NaRbN-TREOR 2 suggested the space groups 1Pna2 and Pnam. The unit-cell volume indicates that Z = 4, so 1Pna2 was chosen, and confirmed by successful solution and refinement of the structure.Crystal data, data collection and structure refinement Fig. 8 details 2HC6H5O7 was solved using Monte Carlo simulated annealing techniques as implemented in EXPO2014 2 was solved using Monte Carlo simulated annealing techniques as implemented in EXPO2014  were carried out using  al. 1994, the bas al. 1991, and the al. 2014. The cal10.1107/S2056989019003190/vn2141sup1.cifCrystal structure: contains datablock(s) KADU1685_publ, kadu1685_DFT, KADU1681_publ, kadu1681_DFT. DOI: Click here for additional data file.10.1107/S2056989019003190/vn2141KADU1685_publsup2.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989019003190/vn2141KADU1681_publsup3.cmlSupporting information file. DOI: 1901423, 1901422, 1901421, 1901420CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Its effects on H2O2-induced cell viability, lactate dehydrogenase (LDH) leakage, intracellular reduced glutathione, and reactive oxygen species (ROS) contents, along with mRNA and protein relative levels of the cytoprotective genes including Nrf2, HO-1, and NQO1, were investigated. The results showed that fucoxanthin could upregulate the mRNA and protein levels of the cytoprotective genes and promote the nuclear translocation of Nrf2, which could be inhibited by the PI3K inhibitor of LY294002. Pretreatment of fucoxanthin resulted in decreased LDH leakage and intracellular ROS content but enhanced intracellular reduced glutathione. Interestingly, pretreatment using fucoxanthin protected against the oxidative damage in a nonconcentration-dependent manner, with fucoxanthin of 5 μM demonstrating the optimal effects. The results suggest that fucoxanthin exerts cytoprotective effects against H2O2-induced oxidative damage in L02 cells, which may be through the PI3K-dependent activation of Nrf2 signaling.Several previous studies have demonstrated the excellent antioxidant activity of fucoxanthin against oxidative stress which is closely related to the pathogenesis of liver diseases. The present work was to investigate whether fucoxanthin could protect human hepatic L02 cells against hydrogen peroxide- (H However, their utilizations have been severely restricted due to their potential toxicity to human health because of their carcinogenic effects . And BHT2O2 or UV-B, with its high antioxidant activity probably attributed to the allenic bond [Carotenoids, natural pigments found in plants, algae, animals, etc., possess the antioxidant ability to protect cells and tissues from detrimental effects caused by ROS . The rolnic bond .Nuclear factor-erythroid 2-related factor 2 (Nrf2 or Nfe2l2) is a transcription factor sensitive to cellular stresses such as oxidative stress. Under normal physiological conditions, Nrf2 is mainly in cytoplasm and transcriptionally inactive due to proteasomal degradation mediated by Kelch-like ECH-associated protein I (Keap1) which acts as an inhibitor. Under oxidative stress, the sulfhydryl groups of Keap1 can be easily oxidized and its binding affinity to Nrf2 decreases, resulting in the translocation of Nrf2 from the cytoplasm to the nucleus and subsequent binding to antioxidant response elements (ARE), which could enhance the expressions of many stress-induced genes such as HO-1 and NQO1 . It has 2O2, UV, and other factors using cell models [2O2, widely used to induce oxidative stress in in vitro models, could easily cross the cellular membranes and generate highly toxic hydroxyl radicals which could react with macromolecules such as DNA and proteins, thus leading to cellular damages [2O2 was similar to that occurred in intact liver [2O2-induced oxidative damage in hepatic L02 cells and whether it works through the Nrf2-ARE pathway.Several studies have reported the antioxidant effect of fucoxanthin, mainly focusing on the scavenging activity against different free radicals  and the ability to quench singlet oxygen –19 usingl models –23. H2O2 damages . As an ect liver . The humct liver , were alct liver . In consμg/mL in a humidified incubator, with CO2 as 5% and temperature as 37°C. Cells were transferred to appropriated multiwall plates and used for subsequent experiments when the confluence reached about 70%. Fucoxanthin  was dissolved in DMSO and diluted with culture medium to various concentrations for following tests. Cells from experimental groups were incubated with fucoxanthin for 2 h prior to the oxidative stress induced by H2O2.Normal human hepatic cell line of L02, purchased from the Procell Life Science & Technology Co., Ltd. , was cultured in RPMI 1640 medium  containing 10% (v/v) fetal bovine serum (FBS), penicillin of 100 U/mL, and streptomycin of 100 5 cells/well, and cultured in serum-free medium for 24 h. Then the cells from experimental groups were pretreated with fucoxanthin at different concentrations  or vitamin E  and cultured for 2 h, followed by exposure to H2O2 . After incubation for 24 h, MTT  was added into each well and incubation for another 4 h at 37°C was performed. Subsequently, the plate was centrifuged at a speed of 800 g for 5 min and the supernatant was discarded. Then the formazan crystals formed in each well were dissolved using 100 μL DMSO and the absorbance was measured using a microplate reader  at a wavelength of 540 nm. The relative cell viability was calculated by comparison with the absorbance of untreated control group.Cell viability of L02 was evaluated through MTT assay. Briefly, cells were seeded into 96-well plates, with cell concentration as 1 × 105 cells/mL) were seeded into 96-well plates and cultured for 24 h. Then cells from experimental groups were subjected to various aforementioned concentrations of fucoxanthin or vitamin E  and cultured for 24 h, followed by treatment of 200 μM H2O2  for 30 min at 37°C. After being washed with PBS, cells were treated with 10 μM DCFH-DA for 20 min at 37°C in a humidified incubator. After the redundant DCFH-DA was removed, cells were washed carefully using PBS three times and the fluorescence intensities of DCF were determined using a fluorescence microplate reader , with excitation wavelength at 485 nm and emission wavelength at 535 nm. And the representative images concerning the levels of intracellular ROS were also captured using an inverted fluorescence microscope  and the fluorescence intensities which reflects the contents of ROS were analyzed using IPP software  and normalized to that of the control group.The intracellular ROS level was evaluated by the 2′,7′-dichlorodihy-drofluorescein diacetate (DCFH-DA) method. Cells (1 × 105 cells/mL) were seeded into 96-well plates and cultured for 24 h. Then the cells from experimental groups were treated with various aforementioned concentrations of fucoxanthin or vitamin E  and cultured for 2 h, followed by treatment of 200 μM H2O2  for another 24 h at 37°C in a humidified incubator. The culture was subsequently mixed with the relevant kit reaction solution and incubated at 25°C. Finally, the LDH activity in culture was determined based on the absorbance at 450 nm using a microplate reader . The LDH leakage rate was calculated by comparing with the maximum LDH activity control group.The LDH leaked in the culture was evaluated using a LDH activity assay kit  according to the supplier's instructions. Briefly, cells . Then the intracellular glutathione was measured using a glutathione determination kit  in accordance with the supplier's instructions. The intracellular glutathione was evaluated based on the absorbance at 420 nm using a fluorescence microplate reader , with excitation wavelength at 350 nm.μM for 1 h, followed by addition of LY294002  and H2O2 of 200 μM . After being treated as aforementioned in method 2.4 and washed three times using PBS, cells derived from each group were collected and the total RNA was extracted using Trizol reagent , followed by the synthesis of cDNA by a commercial first strand cDNA synthesis kit . The relevant cDNA was kept at −70°C before use. The primers, designed using software of Primer5, were synthesized by Takara. Then the triplicate cDNA samples, included in a reaction system of totally 20 μL mainly containing approximately 50 ng cDNA, 10 μM primer for each and 10 μL premix , were analyzed via a real-time quantitative PCR system . The quantitative PCR reaction conditions were as following: 95°C for 2 min, followed by 40 cycles of 90°C for 10 s and 60°C for 40 s. The positive and negative controls were utilized to keep the accuracy of the present quantitative PCR. 2−△△Ct method was used to calculate the relative mRNA levels of target genes, with β-actin as the reference gene. And the sequences of primers used for real-time quantitative PCR in the present study were presented in One additional group named L was added for real-time PCR and the following western blot along with the immunofluorescent assay. As to group L, cells were firstly incubated with fucoxanthin of 5 β-actin was used as internal reference.After being treated as described in method 2.6 and washed using PBS three times, cells derived from each group were collected and lysed using RIPA buffer  containing a protease inhibitor of 1 mM PMSF according to the supplier's instructions. Protein concentrations were determined using BCA method. Equal amounts of proteins were loaded and isolated using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto a PVDF membrane. After being blocked with 5% skim milk, membranes were incubated with primary antibody  and goat anti-rabbit secondary antibody were conjugated to horseradish peroxidase (HRP) (1:200 dilution) . μM for 5 min, followed by being washed using 0.5% PBST three times and adding antifade polyvinylpyrrolidone mounting medium . Then the slices were observed via a fluorescence microscope (Leica DMI400B).After being treated as described in method 2.6, cells were washed using 0.5% PBST and fixed with paraformaldehyde of 4% for 10 min, followed by being washed using 0.5% PBST three times and blocked using immunol staining blocking buffer  for 1.2 h. Then the cells were incubated with Nrf2 rabbit anti-human primary antibody (1:100 dilution) at 4°C overnight, followed by being kept at room temperature for 1 h and washed using 0.5% PBST three times. The cells were subsequently incubated with FITC labeled goat anti-rabbit secondary antibody (1:200 dilution) at 37°C in a humidified incubator for 1.5 h, followed by being washed using 0.5% PBST three times. Finally, cells were stained with Hoechst 33258 of 20  t-test. All the statistical analyses were performed using SPSS software . And it was considered as statistically significant in the case of P value smaller than 0.05.All experiments in the present work were replicated at least three times. All data were presented as means ± SD and analyzed by one-way ANOVA and2O2 at different concentrations  and the appropriate concentration used to induce oxidative damage was evaluated by MTT assay. As is shown in 2O2 was equal to or more than 100 μM . In the presence of 200 μM H2O2, the cell viability demonstrated a moderate decrease. Therefore, treating the L02 cells with H2O2 at a final concentration of 200 μM was selected for subsequent experiments.L02 cells were treated with H2O2-treated L02 cells were also evaluated by MTT. The results were shown in μM vitamin E (VE) or fucoxanthin , while there was no significant difference among the groups (P > 0.05).Effects of fucoxanthin on the viability of H P < 0.01). Compared with model group, VE-treated group exhibited a leakage rate of 18.70 ± 4.98% (P < 0.05), with F5 group demonstrating the lowest LDH leakage (P < 0.05).The leakage level of maximum LDH leakage group was set as 100% and results were presented in P < 0.01). Treatment of VE (50 μM) resulted in the GSH content of 106.94 ± 5.70% . Pretreatment with aforementioned various concentrations of fucoxanthin resulted in the GSH content of 110.69 ± 4.39%, 120.98 ± 6.72%, 103.97 ± 7.12%, and 96.05 ± 5.59%, respectively .As shown in P < 0.01). Treatment of VE (50 μM) resulted in ROS content of 103.66% ± 15.60% . After being pretreated with various aforementioned concentrations of fucoxanthin, the ROS content dropped to 132.60 ± 16.55%, 96.61 ± 19.72%, 105.48 ± 13.65%, and 110.65 ± 11.00%, respectively . The reduced fluorescence intensities, as is shown in 2O2-treated L02 cells.The intracellular ROS contents were presented as a percentage of control. As shown in 2O2-treated L02 cells were shown in Effects of fucoxanthin on nuclear translocation of Nrf2 in HP < 0.01), 0.85 ± 0.07 (P < 0.05), and 0.83 ± 0.06 (P < 0.01), respectively , 1.15 ± 0.11 and 1.61 ± 0.07 (P < 0.01), along with 0.95 ± 0.04 (P < 0.05), and 1.03 ± 0.08 (P < 0.01), respectively, in comparison with model group.Compared with control group, mRNA relative levels of Nrf2, HO-1 and NQO1 from model group dropped significantly to 0.64 ± 0.06 , 0.95 ± 0.05 (P < 0.01), and 0.91 ± 0.03 (P < 0.05), respectively, compared with group F5 . After being treated with various aforementioned concentrations of fucoxanthin, the relative protein levels of Nrf2 were enhanced to 1.01 + 0.04 (P < 0.05), 1.14 + 0.05 (P < 0.01), and 1.02 + 0.05 (P < 0.05), respectively, compared with model group. The relative protein level of HO-1, dropping significantly in model group when compared with control (P < 0.05), was presented in μM, the relative protein level of HO-1 was enhanced to 1.06 ± 0.04 (P < 0.01) and 1.23 ± 0.07 (P < 0.01), respectively, compared with model group. As to the relative protein level of NQO1, significant difference was only found between group F5 and model group (P < 0.01) (As is shown in  < 0.01) .P < 0.01), 0.86 ± 0.07 (P < 0.01) and 0.70 ± 0.13 (P < 0.01), respectively, compared with group F5.After being treated by LY294002, the relative protein levels of Nrf2, HO-1 and NQO1 decreased to 0.87 ± 0.05  [β-carotene and lycopene, also presented lower antioxidant or antitumor activities in vitro at concentrations higher than 10 μM [μM and 20 μM) in the present work might be able to cause the L02 cell membrane more permeabilized in a similar manner to β-carotene [μM might exceed the capability of L02 cells to incorporate fucoxanthin, just like β-carotene utilized in human colon adenocarcinoma cell lines [μM) might play a role of pro-oxidant instead of antioxidant, and its pro-oxidant activity may arise from the 5,6-monoepoxide which has been shown to undergo ring opening reactions resulting from attacking nucleophiles [It must be noted that pretreatment by 5 e (Vero) . Similaran 10 μM , 60. Caoan 10 μM . It has an 10 μM , 63. Thecarotene , consistll lines . Thirdlyeophiles . However2O2-induced oxidative damage in L02 cells. However, data from the present study should be interpreted with caution since there is a limitation that only the cell experiments were performed. Furthermore, it has been reported that many compounds demonstrated lower antioxidant and disease-prevention activities in vivo than they did in vitro, which is called as “the antioxidant paradox” [2O2-induced oxidative damage demonstrated by fucoxanthin suggest that it might be a promising agent in protecting against oxidative stress-related diseases in future.In general, fucoxanthin demonstrated excellent antioxidant activity in defending against the Hparadox” . Therefo2O2-induced oxidative damage, which may be through PI3K-dependent activation of Nrf2 signaling.In conclusion, the results in the present work suggest that fucoxanthin exerts its cytoprotective effects in L02 cells against H


A structural study of the fission yeast Mis19 protein, required for centromere establishment, is presented together with data describing its interactions with functional partners Mis16 and Mis18. CENP-A. Cell cycle–specific deposition of Cnp1 requires the Mis16–Mis18–Mis19 complex, which is thought to direct recruitment of Scm3-chaperoned Cnp1/histone H4 dimers to DNA. Here, we present the structure of the essential Mis18 partner protein Mis19 and describe its interaction with Mis16, revealing a bipartite-binding site. We provide data on the stoichiometry and overall architecture of the complex and provide detailed insights into the Mis18–Mis19 interface.Centromeric chromatin in fission yeast is distinguished by the presence of nucleosomes containing the histone H3 variant Cnp1 Schizosaccharomyces pombe)  . CENP-A–s pombe) . The mecs pombe) . A key ps pombe) . Mis18 is pombe) . In humas pombe) . In fisss pombe) . Bindings pombe) , whereass pombe)  Fig 1A)Schizosacs pombe) . CurrentS. pombe Mis16 both alone and with the N terminus (residues 14–44) of histone H4 bound at 1.9 and 1.8 Å resolution, respectively  that was capable of interaction with Mis16. The crystal structure of Mis16–Mis19C was solved at 2.0 Å by molecular replacement using Mis16 as a search model . Mis19C The C-terminal interaction of Mis19 with Mis16 (site C in our nomenclature) has been previously described . Our cryInterestingly, construct 3, which lacks binding site C, is still able to interact with Mis16, although rather more weakly than the full-length protein as judged by band intensities . Taken tRbbp4 and Suz12 in the polycomb repressor complex 2 (PRC2) complex has recently been reported , whereas the reverse experiment showed that H4α1 could not displace Mis19α2 at any concentration tested. The ITC experiments show that formation of the Mis16–Mis19 complex effectively prevents histone H4 binding to Mis16.Our structures of Mis16–H4α1 and Mis16–Mis19C show that the C-terminal α helix of Mis19C (Mis19α2) binds the same site on Mis16 as the histone H4α1 helix . To bett studies . Interese second . The resTo confirm that Mis19C binding prevents H4 interaction with Mis16 not only for the H4α1 construct but also for the full-length histone, we performed pull-down experiments using a histone H4–Cnp1 dimer  and S4A.D ∼ 51 and 301 nM, respectively, consistent with survival of the F102S mutant at permissive temperature), whereas the R105A mutation substantially reduced binding (KD not determined).To further understand the basis for Mis19 binding to Mis16 at site C, we analysed the contribution of three conserved residues in Mis19α2 that seem to predominantly mediate the interaction. V101 and F102 contribute to a hydrophobic interaction with the wall of the Mis16-binding pocket, whereas R105 forms hydrogen bonds to the side-chain hydroxyl of Mis16 S371 and backbone carbonyl group of Mis16 G370 . Simulta2:Mis192 tetramer. It is possible that this tetramer is similar to the one observed in the crystal structure  . The domom Mis18 .27-C and Mis1944-C). We first asked if these Mis16–Mis19 constructs could still interact with Mis18 in an affinity pull-down experiment (44–112–containing complex (lane 7). Interestingly, however, we do see an interaction with the Mis1927–112–containing construct (lane 5), although the Mis18 band seems somewhat weaker than in the full-length complex  and LxxF (motif 2)  of Mis18 . We trieperiment  and S6A.ractions . This wamotif 2) . The twomotif 2) . Consistmotif 2)  or pull-motif 2) . CollectHere, we describe the architecture of the fission yeast Mis18 complex. The general arrangement involves Mis19 acting as a flexible linker between Mis16 and Mis18. We confirm previous observations that the N terminus of Mis19 binds the C terminus of Mis18, whereas the C terminus of Mis19 binds Mis16 . As prevOur crystal structure, solved using a longer Mis19 construct than that previously studied, shows that there are two additional areas of contact between Mis16 and Mis19, which we call the “A” and “B” sites. The A site is created by the formation of a β-strand in Mis19 completing one propeller blade of the Mis16 structure. This mode of interaction may be common among the RbAp46/48 family and has been exploited in the context of the PRC2, where a so-called WDB2 domain in Suz12 forms a similar interaction with RbAp46. In this case, the interaction also seems to inhibit the binding of H3K4 to the complex . The A s1–Mis191 stoichiometry. Binding studies suggest that both A and C sites can mediate Mis16–Mis19 interaction, as assessed by their individual deletions. In the case of a single Mis16–Mis19 heterodimer, it is conceivable that a single Mis19 molecule could bind a single Mis16 molecule using two sites on Mis19, if site C were to “loop back” to contact Mis16 , 10% glycerol, and protease inhibitors cocktail set III . After clarification by centrifugation, lysate was filtered through a 5-μm filter before chromatography. Proteins were purified using a 5-ml Strep-Tactin Superflow column (QIAGEN). Unbound proteins were washed out with lysis buffer, and the column was washed with buffer B  before elution with buffer B supplemented with 2.5 mM D-desthiobiotin. The eluate was applied onto a Poros HQ anion-exchange column (Applied Biosystems) and eluted with buffer C . The N-terminal tag of Mis16 was cleaved off with TEV protease . Protein was concentrated and applied to a Superdex S75 16/60 (Mis16) or S200 10/300GL (complexes) size-exclusion column  pre-equilibrated with buffer D . Fractions containing the complex were concentrated and either snap frozen in liquid nitrogen or used directly in crystallisation trials. Recombinant S. pombe centromeric histone octamers were formed by co-expression in Escherichia coli and purified using previously described procedures . Single protein constructs were cloned in pFastBac vectors (Invitrogen), whereas multi-protein complexes were cloned using the MultiBac system . The Misocedures .2, 0.8 M NaH2PO4, and 1.2 M KH2PO4, pH 4.5. Crystals of Mis16–H414–44 were grown in 0.05 M LiSO4, 0.8 M NaH2PO4, 1.2 M K2HPO4, and 0.1 M CAPS, pH 9.5. Crystals of Mis16–Mis19C were grown in 0.6 M NaBr, 0.1 M Tris, pH 9.0, and 20% PEG 3350. Crystals were flash frozen and stored in liquid nitrogen. Data were collected under cryogenic conditions (100K) at Swiss Light Source beamline PXIII, ESRF beamline ID23, and Diamond beamline I04. Data sets were integrated and scaled using the Xia2-DIALS pipeline (3CFS) as a search model. The refined Mis16 structure was subsequently used as a search model in molecular replacement solutions of the Mis16–H4 and Mis16–Mis19 structures. All structures were manually rebuilt using Coot pipeline . The strpipeline  using thing Coot , with roing Coot . Data co3/g.The Mis16–Mis18–Mis19 complex was analysed in a buffer containing 150 mM NaCl, 25 mM Tris–HCl, pH 8.0, and 2 mM TCEP. All samples were concentrated to >0.1 mg/ml before loading into cells with identical buffer as a reference. Samples were run for 18 h at 50,000 rpm in an An50-Ti rotor (Beckman Coulter) at 4°C. Data were analysed using SEDFIT , using a27–50 or Mis1997–112 were dissolved in the ITC buffer to a concentration of 260 μM. 3-μl injections were performed at 1,000 rpm stirring speed with an injection spacing of 3 min. The thermodynamic data were processed with the ORIGIN program .ITC experiments were performed at 30°C using an iTC200 . Samples were concentrated in a 50 mM Tris, pH 7.5, 100 mM NaCl, 4% glycerol, and 0.5 TCEP buffer. Protein concentrations were determined using a spectrophotometer. The concentration of protein complex in the ITC cell was 26 μM. The lyophilised peptides of histone H4200 μg of the Mis16–Mis19C complex was injected onto a Superose 6 Increase 10/300 column  coupled to a Wyatt Dawn 8+ MALS system. Data were analysed using Astra software.All in vitro pull-down assays were performed using purified proteins or protein complexes. A total of 30 μl of streptavidin–agarose beads (Thermo Fisher Scientific Pierce) per reaction were equilibrated in binding buffer containing 25 mM Tris–HCL, pH 7.5, 500 mM NaCl, 5% glycerol, and 1 mM TCEP. Strep-Tactin–tagged and untagged proteins were added to the equilibrated beads and incubated for at least 1 h at 4°C on a tube rotation wheel. Tagged bait proteins were at a concentration of 10–20 μM with untagged targets present in a twofold to fivefold molar excess. The beads were washed three times with 500 μl binding buffer and eluted from the beads with binding buffer supplemented by 25 mM desthiobiotin. Input and pull-down elution samples were analysed by SDS–PAGE.6S1L, 6S1R, and 6S29, respectively.Atomic coordinates and structure factors for Mis16, Mis16–H4, and Mis16–Mis19C have been deposited in the Protein Data Bank under accession codes