REST was depleted from the nucleus in prion infected brains and taken up by autophagosomes in the cytoplasm, co-localizing with LC3-II. Importantly, downregulation of the Akt–mTOR and at least partially inactivation of LRP6-Wnt-β-catenin signaling pathways correlated with the decreased levels of REST in vivo in the brain of 263K-infected hamsters and in vitro in PrP106-126-treated primary neurons. Overexpression of REST in primary cortical neurons alleviated PrP106-126 peptide-induced neuronal oxidative stress, mitochondrial damage and partly inhibition of the LRP6-Wnt-β-catenin and Akt–mTOR signaling. Based on our findings, a model of REST-mediated neuroprotection in prion infected animals is proposed, with Akt–mTOR and Wnt-β-catenin signaling as the key pathways. REST-mediated neuronal survival signaling could be explored as a viable therapeutic target for prion diseases and related neurodegenerative diseases.Prion diseases are a group of infectious diseases characterized by multiple neuropathological changes, yet the mechanisms that preserve function and protect against prion-associated neurodegeneration are still unclear. We previously reported that the repressor element 1-silencing transcription factor (REST) alleviates neurotoxic prion peptide (PrP106-126)-induced toxicity in primary neurons. Here we confirmed the findings of the  Prion is a protein-conformation-based infectious agent  and causin vivo? Autophagic vacuoles have been observed in the neurons of experimental hamsters with prion diseases, as well as in synapses of human Creutzfeldt-Jakob disease (CJD) and Fatal familial insomnia (FFI) patients  and Laboratory Animal Requirements of Environment and Housing Facilities . The license number related to their research protocol was 20110611–01 and the animal study proposal was approved by The Laboratory Animal Ethical Committee of China Agricultural University, Beijing China. We aimed to minimize the suffering of the animals and to reduce the number of animals studied at minimum.n = 30, every group consisted of 15 animals) were used in the current study. Each animal was housed on a separate cage. Syrian Golden Hamsters were injected intraperitoneally (i.p.) with 75 μl of 10% brain homogenate in phosphate buffered saline obtained from terminally ill hamsters infected with the 263K strain of prion according to previous protocols (The infectious model of prion disease in hamster was described previously . This isrotocols . The anirotocols : (1) norg for 5 min. The supernatants were collected and boiled for 10 min after addition of a loading buffer . The protein concentration of the supernatants was measured using the BCA assay (CWBio). Protein extracts (10 μl) were subjected to SDS-PAGE, and western blotting  and were sonicated for 15 s, and then centrifuged at 20,000 blotting .Sc in brain homogenates of sick animals was measured by a standard assay by determining the resistance of misfolded proteins to degradation by proteinase K. Brain homogenates were incubated in the presence of proteinase K (50 μg/ml) for 1 h with gentle agitation at 37°C. The digestion was stopped by adding electrophoresis sample buffer. Protease-resistant PrPSc was detected by western blotting, as previously described  (1:500) was purchased from Millipore. Rabbit polyclonal anti-LRP6 antibody (C5C7) (#2560) (1:500), rabbit monoclonal anti-p70 S6 Kinase antibody (#2708T) (1:500), rabbit monoclonal anti-p-p70 S6 Kinase antibody (Thr389) (108D2) (1:500), rabbit monoclonal anti-4E-BP1 antibody (53H11) (#9644T) (1:500), rabbit monoclonal anti-p-4E-BP1 antibody (Thr37/46) (236B4) (1:500), rabbit monoclonal anti-p-Akt antibody (Ser473) (D9E) (1:500), rabbit monoclonal anti-PRAS40 antibody (D23C7) (#2691T) (1:500), rabbit monoclonal anti-p-PRAS40 antibody (Thr246) (C77D7) (1:500), rabbit monoclonal anti-mTOR antibody (7C10) (1:500), rabbit monoclonal anti-p-mTOR antibody (Ser2448) (1:500), rabbit monoclonal anti-p-GSK-3β antibody (Ser9) (D85E12) (1:500), rabbit polyclonal anti-p-β-Catenin antibody (Ser552) (1:500), rabbit polyclonal anti-p-β-Catenin antibody (Ser33/37) (1:500), rabbit monoclonal anti-CREB antibody (D76D11) (1:500), rabbit monoclonal anti-p-CREB antibody (Ser133) (87G3) (1:500), rabbit monoclonal anti-eIF2α antibody (D7D3) (#5324T) (1:500), rabbit monoclonal anti-p-eIF2α antibody (Ser51) (D9G8) (1:500) and mouse monoclonal anti-HA tag antibody (6E2) (#2367) (IF 1:100) were purchased from Cell Signaling Technology . Rabbit polyclonal anti-REST antibody (22242-1-AP) (1:200), mouse monoclonal anti-LC3-II antibody (66139-1-lg) (1:200), rabbit polyclonal anti-Synaptophysin antibody (17785-1-AP) (1:200), rabbit polyclonal anti-PSD-95 antibody (20665-1-AP) (1:200), the rabbit polyclonal anti-Akt antibody (10176-2-AP) (1:200), rabbit polyclonal anti-beta-Catenin antibody (51067-2-AP) (1:200), rabbit polyclonal anti-GSK3β antibody (22104-1-AP) (1:200), mouse monoclonal anti-GAPDH antibody (60004-1-lg) (1:1000), and rabbit polyclonal anti-Lamin B1 antibody (12987-1-AP) (1:500) were purchased from Proteintech Biotechnology . Rabbit polyclonal anti-rat β-actin antibody (Cat No.: AP0060) (1:1000), and goat anti-rabbit IgG (H&L)-HRP secondary antibody (Cat No.:BS13278) (1:5000) were purchased from Bioworld Technology . Goat anti-mouse IgG (H&L)-HRP secondary antibody (1:100), TRITC-conjugated AffiniPure Rabbit Anti-Goat IgG (H+L) (ZF-0317) secondary antibody (1:100), Alexa Fluor 594-Conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (ZF-0516) (1:100), Peroxidase-Conjugated Affinipure goat anti-rabbit IgG (H+L) (ZB-2301) (1:5000) and rabbit anti-goat IgG (H+L) (ZB-2306) (1:5000) were purchased from Beijing ZSGB Biotechnology. Donkey anti-rabbit IgG/FITC (bs-0295D-FITC) (1:100) and Donkey Anti-Goat IgG/Cy3 (bs-0294D-Cy3) (1:100) secondary antibodies were purchased from Beijing Biosynthesis Biotechnology. Neuronal Class III β-Tubulin (Tuj1) antibody (AT809) (1:250), and DAPI dihydrochloride and propidium iodide (PI) were purchased from Beyotime Biotechnology . Reagents and apparatus used in immunoblotting assays were purchased from Bio-Rad .The hamster brain tissue was fixed in 10% buffered formalin solution, and paraffin sections (5-μm in thickness) were prepared routinely. Sections were treated with an antigen retrieval kit  for 1 min at 37°C and quenched for endogenous peroxidases in 3% H2O2 in methanol for 10 min. After blocking in 1% normal goat serum, the sections were incubated with 1:100-diluted polyclonal antibody for REST at 4°C overnight. Rabbit isotype antibody  was used as the negative control. Subsequently, the sections were incubated with 1:250-diluted HRP-conjugated goat anti-rabbit secondary antibody  for 60 min, and visualized by incubation with 3,3′-diaminobenzidine tetrahydrochloride (DAB). The slices were counterstained with hematoxylin, dehydrated and mounted on a slide and viewed under an Olympus BX51 microscope. Density analysis was performed using the Image J software . REST staining for each region was separately assessed and the mean optical density (MOD) was recorded. The OD analyses were performed by two investigators in a double-blind manner. Non-specific background correction in each section was done by subtracting the OD value of the blank area obtained from the same section.The PrP peptide PrP106-126 (sequences KTNMKHMAGAAAAGAVVGGLG), was synthesized by Sangon Bio-Tech . The purity of prion peptides was >95% according to the data from the synthesizer. FITC-labeled PrP106-126 peptides were purchased from Fanbo Biochemicals . The peptides were dissolved in 0.1 M PBS to a concentration of 1 mM, and left to aggregate at 37°C for 24 h before each experiment. Experiments were conducted with the final peptide concentration of 200 or 300 μM.5 cells/cm2 on polyethyleneimine (Sigma)-coated plates and cultured in DMEM F12 , supplemented with 2% B27 (Invitrogen) and 0.5% Penicillin-Streptomycin (Gibco). Two days later, 10 μM cytarabine (Sigma) were added to repress the growth of glial cells.Dissociated cerebral cortex neuronal cultures were prepared from postnatal 1-day-old Sprague-Dawley rats, according to the previously described procedure . BrieflyThe pCMV-HA-Rest vector of full-length REST (Cat No. PPL50007-2a) was obtained from the Public Protein/Plasmid Library . The pGPH1/GFP/Neo-REST-Rat short-hairpin RNA (shRNA) against Rat REST were obtained from GenePharma . Mature antisense sequences of effective shRNAs were 5′-GCTGTGGCTACAATACCAACC-3′ (946-966) (shREST-1) and 5′-GTGCAATTATGTGGCCTCTAA-3′ (1295-1315) (shREST-2). For transfection , cultureFor protein localization examination, PCCN grown on cover slips were washed twice with PBS, fixed by Immunol Staining Fix Solution (P0098), blocked 1h at room temperature by Immunol Staining Blocking Buffer (P0102) and then incubated overnight at 4°C with the appropriate primary and secondary antibodies. The nucleus was stained with DAPI. Primary neuronal cells were labeled using murine anti-Tuj1  antibody  and fluorescein isothiocyanate (FITC)-conjugated donkey anti-mouse antibodies as the primary and secondary antibodies, respectively.For the extraction of total cell proteins, primary neurons were washed in PBS after overexpression, knockdown or peptide treatment, and then dissolved and homogenized with pre-chilled RIPA lysis buffer  supplemented with a proteinase inhibitor cocktail . Cell lysates were centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was harvested. The supernatant was frozen at -80°C for western blot analysis of protein expression.Cytoplasmic and nuclei proteins were extracted using a cytoplasmic and nuclear protein extraction kit (Wuhan Boster Biotech) and subjected by western blotting. The blot was stripped and probed with anti-β-actin (for cytoplasmic extracts) or anti-Lamin B (for nuclear extracts) antibodies.Extracted proteins were separated by SDS-PAGE on 10–15% gels, and then transferred onto a nitrocellulose membrane. Non-specific binding sites were blocked by 5% dried fat-free milk in Tris-buffered saline . Primary antibodies were added and incubated at 4°C overnight as described previously. Membranes were washed with TBS-T, and then incubated with the secondary antibody, either goat anti-mouse IgG or anti-rabbit IgG horseradish peroxidase-conjugated antibody (1:5000). Bands of immunoreactive proteins were visualized on an image system  after membrane incubation with enhanced chemifluorescence (ECF) reagent for 5 min as described previously.Reactive oxygen species were measured using the non-fluorescent probe 2′7′-dichlorofluorescein diacetate . DCFH-DA passively diffuses into primary neurons and is deacetylated by esterases to form non-fluorescent 2′7′-dichlorofluorescein (DCFH). In the presence of ROS, DCFH reacts with ROS to form the fluorescent product DCF, which is trapped inside cells. Briefly, the culture medium was first removed and the cells were washed with PBS before incubation in DCFH-DA, diluted to a final concentration of 10 μM, for 20 min at 37°C. ROS levels were assessed by measuring the fluorescence at an excitation of 485 nm and an emission of 528 nm .2. The absorbance of the samples was measured at 450 nm using a microplate reader with a background control as the blank. The cell survival ratio was expressed as the percentage of the control group . Briefly, after each treatment, 10 mm of WST-8 reagent was added and primary neurons were incubated for 2 h at 37°C and 5% COol group .To analyze mitochondrial distribution, treated-PCCN cells were incubated with pre-warmed (37°C) staining solution containing the Mitotracker Green FM probe (M7514) (Invitrogen)  for 45 min. After staining is complete, the staining solution was replaced with prewarmed fresh media and observed using an upright fluorescence confocal microscope . The number of cells with non-tubular/fragmented mitochondria was determined, with at least 500 transfected cells examined. All quantifications were based on at least three independent experiments.4 in 0.1 M sodium cacodylate buffer on ice for 1 h and dehydrated with acetone. The cell pellet was embedded in EM-bed 812 resin and polymerized at 60°C for 48 h. Ultrathin sections (70 nm) were obtained on a Leica Ultracut UCT ultramicrotome  and counterstained with uranyl acetate and lead citrate before observation under a JEM-2100 TEM .Primary neurons were treated and then washed twice in PBS, trypsinized, fixed in ice-cold 5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) at 4°C for 15 min, and then centrifuged. Cell pellets were fixed for 4 h. After a complete rinse with sodium cacodylate buffer, the cell pellet was further fixed in 1% OsOpost hoc Turkey’s test using the SPSS software , GraphPad Prism 5 software  and Image J software . P < 0.05 was considered statistically significant.All assays were performed on three separate occasions. Data were expressed as means ± SD. We have checked the distribution of all datasets and all were parametric. All comparisons for parametric data were made using Student’s test or one-way ANOVA followed by Figure 1A) firstly . In line with a previous in vitro study  and stained with haematoxylin and eosin (H&E)  for observing and confirming brain lesions induced by 263K scrapie  . In the control animals, the pattern of REST immunoreactivity was evenly distributed in both the cytoplasm and nucleus of all regions of the brains  . However, low expression of REST was observed in the cortex and medulla oblongata of 263K-infected hamsters, especially in the nucleus  . The marked down-regulation of REST in the 263K-infected hamsters in in vivo and in vitro experiments  or cortex slices of scrapie-infected hamsters since those regions showed a significant alteration of REST in the previous data. Additionally, subcellular localization of REST in the nucleus and cytoplasm fractions of isolated cortex, medulla oblongata, cerebellum, and hippocampus of normal control and 263K-infected hamsters were quantified by western blotting  using GAPDH and Lamin B as the cytoplasmic and nuclear marker, respectively . As expected, in agreement with the IHC results, REST was sparsely distributed in the nucleus of cortex and medulla in the 263K-infected hamsters compared to the normal control . Cytoplasmic levels of REST were low and comparable between the infected and control groups (exclude cortex) . The relatively increased level of REST in the cortex of 263K-infected hamsters compared with the normal control in line with our previous in vitro experiments  consistent with the data shown in Figures E.Furthermore, previously, autophagic vacuolation and hyperactivation of the autophagic system in neurons of prion diseases were observed under electron microscope . We prevFigures E). In particular, p-Akt (Ser473) and p-mTOR (Ser2448), biochemical indicators of Akt and mTOR activation .The Akt-mTOR pathway is an important negative signal for autophagy in mammalian cells . The mactivation , were deFigure 4C) were observed in the normal group but disappeared in the 263K infected group. This in vivo experiment indirectly confirms our previous data that LRP6-mediated Wnt-β-catenin signaling partly regulates the expression of REST . β-catenin is a direct substrate of GSK-3β and an intermediate effector of the canonical Wnt signaling pathway  , which s of REST . β-caten of REST  and GSK3 of REST , as the  of REST , while t of REST . Interes pathway . GSK-3 a pathway . The dat pathway , a signiin vitro study of prion diseases, neurotoxic prion peptide (PrP106-126) is used as a material in our further research in vitro in PCCN. PrP106-126-induced neurotoxicity and pathological damage in PCCN had been proved in our previous studies  with prolonged response time. Cell viability in different treated group was detected using the CCK-8 assay and treatment with staurosporine (500 nm for 24 h) (P < 0.01); (2). Consistent with our previous data (P < 0.01) and shREST (52.6% viability) (P < 0.01) groups, REST-overexpression significantly increased cell viability (78.3% viability) (P < 0.001) .As a widely used model for the  studies . Here, wor 24 h)  as a posous data , after tin vivo data in infected hamsters, total mTOR protein and p-mTOR (Ser2448)/total mTOR protein declined with prolonged stimulation by the prion peptide compared with the PBS control group (P < 0.001) ; Although levels of total Akt protein had no significant change , p-Akt (Ser473)/total Akt protein showed a transient increase after incubation for 6–12 h and then declined with prolonged stimulation compared with PBS control group . Furthermore, total and phosphorylated forms of proteins downstream of Akt-mTOR, including total PRAS40 protein, p-PRAS40 (Thr246), total p70 S6K protein, p-p70 S6K (Thr389), total 4EB-P1 protein and p-4E-BP1 (Thr37/46), were examined. All the total forms of these proteins had no significant change . Notably, the phosphorylated forms of these proteins  had a tendency to decrease, demonstrating strong inhibition of the Akt-mTOR pathway by the neurotoxic prion peptide .Next, to further explore the potential relationship between REST and dysfunctional regulatory pathways, we also determined if the Akt-mTOR and LRP6-Wnt-β-catenin signaling pathways were altered in PCCN after exposure to PrP106-126. PCCNs were treated with 200 μM PrP106-126, and the protein extract at various time points (0–48 h) was analyzed by western blotting. Consistent with the Figures 6A,D) in line with our previous in vitro studies (P < 0.001) . The different outcomes of normalized p-β-catenin (Ser552) in vivo and in vitro might due to the different treated time in each prion diseases models and varying response of molecules to the course of diseases. p-β-catenin (Ser33)/total β-catenin protein was significantly induced by the peptide (P < 0.001) , indicating β-catenin destabilization (P < 0.001) . The above data demonstrated the partially inactivation of LRP6-mediated Wnt-β-catenin signaling pathway in prion peptide-treated neuronal cells, consistent with the in vivo findings in 263K-infected hamsters and results of our previous study . Total and phosphorylated forms of CRE-binding protein (CREB) were tested as CREB plays a key role in promoting neuronal survival and axon extension , level of total eIF2α protein had no significant change  while p-eIF2α (Ser51)/total eIF2α protein was significantly induced by PrP106-126 over time .We previously showed that PrP106-126 induced caspase-3 activation. Here, Poly (ADP-ribose) Polymerase (PARP), one of the main cleavage targets of caspase-3, was examined. PARP maintains cell viability, and cleavage of PARP facilitates cellular disassembly and serves as a marker of apoptosis . PARP inxtension . Total axtension  was alsoIn summary, the neurotoxic prion peptide, PrP106-126 inhibited the Akt-mTOR and Wnt-β-catenin signaling pathways, associated with the downregulation of REST, and disrupted the neuronal survival pathway by promoting apoptosis and stress in PCCN.Figures C). Examined total and phosphorylation of β-catenin and GSK3β to evaluate the effect of REST on the Wnt-β-catenin pathway . The results demonstrated that, under the stimulation of PrP106-126, overexpression of REST: (1) enhanced p-mTOR (Ser2448)/total mTOR protein (by 2.84-fold relative to the HA-vector group), p-β-catenin (Ser552)/total β-catenin protein (by 3.80-fold relative to the HA-vector group) and p-GSK3β (Ser9)/total GSK3β protein (by 2.49-fold relative to the HA-vector group); (2) alleviated PrP106-126-induced decrease of both total form and phosphorylation (Ser133) form of CREB by 1.30- and 1.41-fold, relative to the HA-vector group, respectively. (3) overexpression of REST significant restored the levels of mTOR, p-mTOR (Ser2448), p-β-catenin (Ser552), total and phosphorylation (Ser9) forms of GSK3β compared with the knockdown of REST group. Conversely, under the PrP106-126-induced pathological conditions, knockdown of REST exacerbated the inactivation of these proteins  compared with the HA-vector group, further suggesting the important and essential role of REST in the regulation of Akt-mTOR and Wnt-β-catenin signaling pathways in neuronal cells.We overexpressed or blocked REST as described before  and thenFigures 8A,B). Consistent with the western blotting results, REST significantly prevented the inhibition of the phosphorylation of Akt, mTOR , β-catenin and GSK3β in PCCN by PrP106-126 . Compared with the negative control (set as 100%), the relative arbitrary fluorescence units (AFU) of p-Akt (Ser473), p-β-catenin (Ser522) and p-GSK3β (Ser9) were markedly suppressed by 56, 62, 60, and 72%, respectively, by PrP106-126. Overexpression of REST recovered the levels of phosphorylated Akt (78% of the negative control), mTOR (115%), β-catenin (96%), GSK3β (178%), thereby alleviating PrP106-126-mediated inactivation of the Akt-mTOR and Wnt-β-catenin pathways.To further directly observe and confirm the correlation of REST expression and Akt–mTOR and Wnt-β-catenin pathways in the prion disease model, p-Akt (Ser473), p-mTOR (Ser2448), p-β-catenin (Ser522), and p-GSK3β (Ser9) levels were quantified in PrP106-126-treated PCCN by immunofluorescence and expressed as percent of the HA-vector-transfected, untreated negative control. Firstly, confocal immunofluorescence labeling for total REST (green), exogenously expressed REST (Red) (stained with HA antibody) and nucleus  was quantitative analysis to show the transfection efficiency of exogenous REST in PCCN . The location and expression of p-CREB (Ser133) was further observed by immunofluorescence. As expected, REST overexpression significantly increased p-CREB to 135% from 34.1% in the group without REST transfection . As our previous reports showed that REST enhanced the expression of transcription factor FOXO1, an anti-apoptotic protein, which also mediates oxidative stress resistance  fluorescent probe (green). ROS was suppressed by REST overexpression .In consideration of the function of both CREB and REST in the synaptic plasticity and neuroprotection , increassistance  and is psistance , we furtFigures 10C,F). Combining with our previous data that overexpression of REST restored mitochondrial function and alleviated PrP106-126-induced abnormal mitochondrial structures  is necessary for the maturation and cell surface location of this receptor .Our data illustrates that REST regulates neuron survival and is a critical neuroprotective factor in prion diseases and possibly other neurodegenerative disorders. The signally pathways for REST and associated molecules in neurodegenerative disorders are proposed (Conceived and designed the experiments: ZS. Performed the experiments: ZS, SS, WY, and HD. Analyzed the data: ZS. Wrote the paper: ZS. Edited the paper: ZS, SS, LY, XZ, and DZ. Contributed reagents/materials/analysis tools: DZ. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Theba pisana, a hermaphroditic land snail that has become a highly invasive pest species within agricultural areas throughout the world. Our analysis of T. pisana CNS tissue has revealed gene transcripts encoding molluscan reproduction-associated proteins including APGWamide, gonadotropin-releasing hormone (GnRH) and an egg-laying hormone (ELH). ELH isoform 1 (ELH1) is known to be a potent reproductive peptide hormone involved in ovulation and egg-laying in some aquatic molluscs. Two other non-CNS ELH isoforms were also present in T. pisana (Tpi-ELH2 and Tpi-ELH3) within the snail dart sac and mucous glands. Bioactivity of a synthetic ELH1 on sexually mature T. pisana was confirmed through bioassay, with snails showing ELH1-induced egg-laying behaviours, including soil burrowing and oviposition. In summary, this study presents a detailed molecular analysis of reproductive neuropeptide genes in a land snail and provides a foundation for understanding ELH function.Increased understanding of the molecular components involved in reproduction may assist in understanding the evolutionary adaptations used by animals, including hermaphrodites, to produce offspring and retain a continuation of their lineage. In this study, we focus on the Mediterranean snail,  Snail reproduction is controlled by a tetra neural central nervous system (CNS) ,2 that cet al. 1+ = 1610.83) . Three different Tpi-ELH1 precursor peptide segments were identified from the mucous gland extracts, which also eluted within 30–35 min , an example of which corresponds to Q43LEITGKQP51, ([M+2H]2+ = 507.28).RP-HPLC was performed on CNS and mucous gland extracts to identify whether any of the T. pisana were also analysed for egg-laying following injection of snails with either synthetic Tpi-ELH1 or control solutions . In Assay 1, within 60 min post-injection 100% of snails (n = 78) had recovered, as demonstrated by normal activity. No behaviour change, including egg-laying, was observed in negative controls  24 h post-injection. By contrast, soil-burrowing behaviour was observed in 40.7% of snails treated with Tpi-ELH1 (n = 22), including a single Tpi-ELH1-treated snail (1.15x10-4M Tpi-ELH) that had produced an egg mass containing fertilised and non-fertilised eggs. In subsequent observations at 48 h and 72 h post-injection, an additional 5 snails from treatment group ELH 1.15x10-4 M had laid egg cordons. No mortality was observed in any of the treatment groups.Mature -4M Tpi-ELH showed egg-laying, in Assay 2 snails (n = 30) were injected with only 1.15x10-4 M ELH . Again, no immediate egg-laying was observed, but by 24 h post-injection, 3 snails had exhibited egg-laying behaviour (burrowed into the soil), and 2 snails had laid eggs. After 48 h post-injection, another 13 snails had laid eggs, while 2 more egg masses were present at 72 h post-injection. Some egg masses were found above the top soil (n = 3). By day 6, 2 more snails produced egg masses before the experiment was terminated. Negative control  produced no eggs or and snails did not exhibit burrowing or egg-laying behaviour.Given that snails given 1.15x10T. pisana to assess the presence of molluscan-like reproductive neuropeptide genes. Our T. pisana CNS RNA-seq data revealed the presence of two partial-length Tpi-APGW1 precursor isoforms (Tpi-APGW1 and Tpi-APGW2). At 379 amino acids, and encoding twelve APGWamide peptides, Tpi-APGW1 was larger (by at least 72%) than the Haliotis asinina APGWamide precursor  EAERDRRTWSISNALTVLTDMVVEHEQRRLAAEREALKQRLLELamide.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Myelodysplastic syndromes (MDS) are a diverse group of malignant clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, dysplastic cell morphology in one or more hematopoietic lineages, and a risk of progression to acute myeloid leukemia (AML). Approximately 50% of MDS patients respond to current FDA-approved drug therapies but a majority of responders relapse within 2-3 years. There is therefore a compelling need to identify potential new therapies for MDS treatment. We utilized the MDS-L cell line to investigate the anticancer potential and mechanisms of action of a plant-derived compound, Withaferin A (WFA), in MDS. WFA was potently cytotoxic to MDS-L cells but had no significant effect on the viability of normal human primary bone marrow cells. WFA also significantly reduced engraftment of MDS-L cells in a xenotransplantation model. Through transcriptome analysis, we identified reactive oxygen species (ROS)-activated JNK/AP-1 signaling as a major pathway mediating apoptosis of MDS-L cells by WFA. We conclude that the molecular mechanism mediating selective cytotoxicity of WFA on MDS-L cells is strongly associated with induction of ROS. Therefore, pharmacologic manipulation of redox biology could be exploited as a selective therapeutic target in MDS. Myelodysplastic syndromes (MDS) are a diverse group of malignant clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, dysplastic cell morphology in one or more hematopoietic lineages, and a risk of progression to acute myeloid leukemia (AML) , 2. InefMDS is incurable with current FDA-approved drug therapies . Approximately 50% of MDS patients respond to these therapies and a majority of responders relapse within 2-3 years –8. PatieWithania somnifera  with demonstrated anticancer activities in several cancer models including prostate, breast, cervical and pancreatic cancers, as well as melanoma and lymphoma [Scientific exploration of plant-derived compounds for cancer treatment is increasing because they are thought to be less toxic than current chemotherapy and radiotherapy regimens . Withafelymphoma –15.in vitro and in vivo. Biochemical studies identified reactive oxygen species (ROS) induced JNK/AP-1 apoptotic cell death as a major pathway by which WFA causes cytotoxicity in MDS-L cells.The heterogeneity of MDS has made it difficult to generate a mouse that models complete disease phenotype, and xenotransplantation of patient bone marrow cells into immunocompromised mice is poor and highly inefficient , 17. We in vitro model for MDS but is also representative of highly aggressive disease, displaying deletions in chromosomes 5 and 7 [in vitro proliferation of MDS-L cells in a dose dependent manner  xenograft model  to invested mice . WFA  regulated by WFA in MDS-L cells, and focused on early time points (6 and 12 h) to identify primary gene alterations. Shown in During apoptosis, a decline in MMP ultimately results in the activation of caspases by proteolytic cleavage, a hallmark of apoptosis , 29. Of JUN and FOSB were among the top 3 up-regulated genes at 6 h (47 and 36 fold respectively) and the top 2 up-regulated genes at 12 h (52 and 60 fold respectively). C-JUN and FOSB heterodimerize to form an AP-1 transcription factor that is activated by phosphorylation of the C-JUN subunit by JNK, a signaling pathway that has been demonstrated to regulate apoptosis [We next sought to identify the critical pathway(s) mediating the anti-proliferative effects of WFA on MDS-L cells. The finding that growth suppression of MDS-L cells by WFA involved cell death by apoptosis Figure  narrowedpoptosis . A receppoptosis . Since R2DCFDA , mediates JNK signaling by phosphorylating and activating MKK7 , 32. TheWe next investigated the potential functional relationship between increased C-JUN phosphorylation and the transcriptional activity of AP-1. MDS-L cells transfected with either an AP-1 reporter or the empty vector expressing firefly luciferase were treated with WFA for 12 h, and promoter activity assessed by the dual Glo luciferase assay. The increase in C-JUN phosphorylation led to an increase in AP-1 activity since WFA-induced AP-1 promoter driven luciferase activity in MDS-L cells was comparable to phorbol myrsitate acetate (PMA) Figure , the knoTo address the importance of JNK signaling in WFA-induced apoptosis of MDS-L cells, we employed a well-characterized selective covalent JNK inhibitor , JNK-IN-Off-target effects are always a concern with the use of small-molecule inhibitors in biological systems. To address this issue, we used another widely used reversible ATP-competitive JNK inhibitor, SP600125 . Similarin vitro and in vivo.The significant contribution of JNK/AP-1 signaling in apoptotic cell death of MDS-L cells was corroborated by a reduction in annexin-V/PI positive cells upon JNK inhibition with JNK-IN-8 in WFA-treated cells Figure . InteresWithania somnifera, WFA, is selectively cytotoxic to MDS-L cells both in vitro and in vivo. Using gene expression changes induced by WFA and systematic biochemical analysis, we identified ROS-activated JNK/AP-1 signaling as a major pathway through which WFA induces MDS-L apoptosis. The efficiency with which NAC prevented WFA-induced apoptosis of MDS-L cells and the failure of WFA to induce oxidative stress in normal human primary bone marrow cells suggest that the molecular mechanism mediating selective cytotoxicity of WFA on MDS-L cells is associated with induction of ROS. This was supported by the observation that WFA does not induce cell death in normal human primary bone marrow cells but induces apoptosis of MDS patient-derived primary bone marrow cells.The responses to current drug treatments for MDS is poor –8. HenceFor the first time, our studies established a link between WFA-induced ROS, JNK/AP-1 signaling and apoptosis in MDS-L cells. WFA-induced AP-1 activation was demonstrated by an increase in the mRNA expression of bona fide AP-1 targets , 36, BIMRedox homeostasis is an essential albeit delicate balance as excess ROS can result in apoptotic cell death . Most caA major goal in cancer therapy is to develop drugs that selectively target malignant cells with minimum toxicity to normal cells. The implication of oxidative stress in the development and prognosis of MDS  suggestsWithania somnifera extract (Sabinsa Corp) using a series of solvent extractions and silica gel-based vacuum liquid column chromatography at the University of Louisville and at the laboratory of Dr. I. P. Singh, National Institute of Pharmaceutical Education Research (NIPER), India. The purity was found to be >94% by UPLC. WFA was reconstituted in dimethyl sulfoxide (DMSO). Anti-human CD45-PE (12-9459-42) and CD33-FITC (11-0339-42) were purchased from eBioscience . Cremophor (C5135), Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) (C2920), hydrogen peroxide (H2O2) (H1009), Phorbol 12-myristate 13-acetate (PMA) (P1585), N-acetyl-cysteine (NAC) (A8199), RNase A (R6513), SP600125 (S5567), JNK-IN-8 (SML-1246), JC-1 (T4069), HEPES solution (H0887), ethylenediaminetetraacetic acid (EDTA) solution (E7889), dimethyl sulfoxide (D2438) and monoclonal anti-β-actin antibody (A5441) were purchased from MilliporeSigma-Aldrich . Antibodies to P-p38 (9211S), total p38 (9212), cleaved caspase-3 (9661S), total caspase-3 (9665S), P-MKK7 (4171S), P-c-Jun (9261S), total c-Jun (9162), GAPDH (2118S), CDK2 (2546P), cyclin A (4656P), CDK1 (9116) and cyclin B (4135) were obtained from Cell Signaling Technology . Anti-total MKK7 was purchased from Zymed (32-7000). Antibodies to Hdac1 (7872), NF-κB p65 (372) P-JNK (6254) and total JNK (571) and NF-κB p65 (372) were obtained from Santa Cruz Biotechnology . Peroxidase coupled goat anti-rabbit (SC-2004) and anti-mouse (SC-2005) Ig secondary antibodies were also acquired from Santa Cruz Biotechnology . 1X phosphate buffered saline (16750-078) was obtained from VWR . Lenalidomide (NC0600901) was purchased from Thermo Fisher Scientific . Biotin conjugated rat anti-CD45R/B220 (553086), anti-CD11b (553309), anti-Gr-1 (553125), anti-CD8α (5532029), anti-Ter-119 (553672), anti-CD5 (553019); streptavidin APC CY7 (554063) and anti c-KIT-APC (553356) were purchased from BD Pharmingen . Anti-Sca-1-PB (122520) was purchased from BioLegend .WFA was isolated from ™ plus  and maintained in RPMI medium supplemented with 50 μM 2-mercaptoethanol, 1 μM sodium pyruvate and 10% fetal bovine serum (FBS). Human primary cells were obtained in accordance with the Declaration of Helsinki and approval from the institutional review board, protocol # 88-00241. MDS/AML samples were obtained from Leukemia Tissue Bank of the Ohio State University Comprehensive Cancer Center (OSU CCC). MDS/AML cells were thawed and maintained in RPMI medium supplemented with 10% fetal bovine serum and 10 ng/ml hIL-3, GM-CSF and stem cell factor (R&D Systems). All media were purchased from GIBCO-Life Technologies.MDS-L cells were maintained in IMDM/Ham's F-12 (50:50) medium supplemented with 12% fetal bovine serum , 5 μg/ml apotransferrin, 50 μM 2-mercaptoethanol (2-ME) and 20 ng/ml of human IL-3 (PeproTech). The cell line was authenticated by expression of cell surface markers as previously described . Human p5) or normal human primary bone marrow (1 × 106) cells were treated with increasing concentrations of WFA (0 – 20 μM) in 96 well flat-bottom microtiter plates for 48 h in 0.2 ml of media. Cells were cultured in quadruplicates. Treated cells were incubated with 0.5 mg/ml MTT -2, 5-diphenyltetrazolium bromide) dye (Sigma Aldrich) for 4 h followed by solubilization of formazan salt with acidic isopropanol and spectrophotometric measurements at 560 nm and 690 nm. Optical density variation was corrected by subtracting OD 690 from OD 560 nm values. Media background was subtracted from all treatment groups and a DMSO control group was included in each experiment. IC50 was computed by Graphpad analysis.MDS-L (1.5 × 10137Cesium γ-irradiator (J.L Shepherd and Associates). Four hours later, mice were engrafted by intravenous injection with MDS-L cells (1 × 106 cells/mouse).Animal studies were conducted under an approved protocol (# 2011-0904) by the University of Kentucky Institutional Animal Care and Use Committee. NOD/SCID-IL2R γ-hSCF/hGM-CSF/hIL3 (NSGS) mice were obtained from The Jackson Laboratories . 6 -7 month old male and female littermates were exposed to 2.5Gy irradiation in a Mark I-68 +/CD33+ positive cells in the bone marrow compartment. Positively stained cells were detected by the BD LSRII flow cytometer and the data was analyzed by the FlowJo  single cell analysis software.Mice were treated from day 14 post-engraftment with 8 mg/kg of WFA intraperitoneally 5x a week for 6 weeks. Control mice received vehicle , 70% phosphate PBS). MDS-L engraftment was assessed by the percentage of human CD45Bones which had been preserved in formalin (Fisher Scientific #SF93-4) were washed with running tap water for an hour and decalcified in Richard-Allan scientific decalcifying solution (Thermo Scientific #8340) for 3 h. Decalcified bones were washed, cut horizontally, processed, embedded in paraffin, sectioned and routinely stained with hematoxylin and eosin. The slides were evaluated by a veterinarian blinded to group treatments for abnormalities.5 cells/ml) were exposed to WFA (10 μM) or DMSO for 8 h. Cells treated with FCCP (50 μM) for 2 h were used as a positive control. JC-1 was added at a 1 μM final concentration to cells for the last 30 min of treatment at 37°C and fluorescence was measured by the iCyt Synergy sorter system  with 488 and 561 nm lasers. WinList 3d 8.0 software  was used for data analyses. For microscopy, cells treated with WFA (10 μM) or DMSO and stained with JC-1 as described above were mounted on poly-l Lysine (MilliporeSigma-Aldrich # P-6282) coated slides by Cytospin. Pictures were taken on the same day with a Nikon A1RSi confocal microscope .MDS-L cells (7.5 × 105 cells/ml) were cultured with 10 μM WFA or DMSO for different time points. Alternatively, cells were pretreated with JNK-IN-8 (10 μM) or NAC (25 mM) for 4 h before further treatment with WFA (10 μM) for the indicated time points. Cells were lysed in Cell Signaling lysis buffer (#9803) containing 1mM PMSF (Sigma P7626), 2mM NaF (Sigma S-1504), 2mM Na3VO4 (Sigma S-6508) and 1x protease inhibitor cocktail (Roche 5892953001). 35 μg total protein/sample of total lysate was subjected to SDS polyacrylamide gel electrophoresis. Western blot analysis was performed as previously described [MDS-L cells  or NAC (25 mM) pretreatment, or no pretreatment were treated with WFA (10 μM) for 24 h and stained with annexin-V/PI. Stained MDS-L cells were detected by the BD LSRII flow cytometer and BD CellQuest™ Pro software was used for data analyses.The annexin-V apoptosis detection kit (A432) from Leinco Technologies  was used for annexin-V assays. Thawed human primary MDS/AML cells were maintained in culture for 24 h and then treated with increasing concentrations of WFA for 24 h. Treated cells were stained with annexin-V-FITC and PI following the manufacturer's protocol. Data was acquired with the Becton-Dickinson FACSCalibur flow cytometer and analyzed with the BD CellQuest™ Pro software . For MDS-L cells, 7.5 × 106 MDS-L cells were treated with WFA (10 μM) or DMSO for 6 h or 12 h and total RNA was extracted using the Direct-zol™ RNA miniprep kit (Zymo Research #R2051). RNA purity was assessed using the Agilent RNA 6000 Nano assay kit (#5067-1511) on the Agilent 2100 Bioanalyzer . The RNA integrity number (RIN) was ≥ 9 for all samples. Sense-strand DNA (ss-cDNA) was generated, amplified and biotinylated using the WT Plus Reagent kit  from 100 ng total RNA per sample. 30 μg of fragmented biotin-labelled ss-cDNA was hybridized to Affymetrix human gene 2.0 ST arrays at 45°C and 60 rpm for 16 h. The arrays were washed, stained using the Affymetrix fluidics station FS 450 and scanned on the Affymetrix 7G GeneChip Scanner. The raw microarray data files were processed through Oligo [10 × 10gh Oligo  for datalimma [Gene expression profiles of MDS-L cells were examined in triplicate using Affymetrix human gene 2.0 ST arrays. Differential expression analyses comparing WFA-treated and control groups were performed by limma . Signifilimma .R reagent (LifeTechnologies #15596-018) and cDNA was synthesized from 500 ng of total RNA with qScript reverse transcriptase (Quanta Biosciences #95048-100). iTaq™ universal SYBRR green fluorescent Supermix (Biorad #172-5121) was used to quantify mRNA expression. RNA polymerase II was used as an internal control. BIO-RAD CFX Manager software was used to perform relative quantification of target genes using the comparative CT (ΔΔCT) method. Primers were obtained from Integrated DNA Technologies  and the sequences are provided in Total RNA was extracted by TRIzol2DCFDA) (ThermoFisher Scientific #C400), was used as an indicator for intracellular ROS measurement. MDS-L cells were treated with DMSO, WFA (10 μM) or H2O2 (1 mM) for 30 min at 37°C. Alternatively, cells were treated with N-acetyl-cysteine (NAC) (25 or 50 mM) for 4 h followed by DMSO, WFA (10 μM) or H2O2 (1 mM) for additional 30 min at 37°C. Treated cells were suspended in warm H2DCFDA solution (1.25 μg/ml in PBS) and incubated in the dark at 37°C for 20 min. Fluorescence was detected on the BD LSR II flow cytometer and the BD CellQuest™ Pro software was used for data analyses. The oxidized form of DCFDA, 5-(and-6)-Carboxy-2', 7'-Dichlorofluorescein Diacetate (Carboxy-DCFDA) (ThermoFisher Scientific #C369), was used as a control for uptake, cellular esterase activity and decay.Cell permeant 6-Carboxy-2', 7'-Dichlorodihydrofluorescein Diacetate (Carboxy-H6 cells were co-transfected with 20 μg of TK Renilla luciferase vector and 40 μg of firefly luciferase pGL3 vector (AP-1 or empty vector) at 250 mV, 960 μF and 200 Ω in 200 μl of MDS-L culture medium with a Gene Pulser electroporator . Transfected cells were cultured in MDS-L culture medium for 24 h at 8.5 × 105 cells per well in 6 well flat bottom plates (Corning #353224). 24 h after transfection, 1 × 105 cells per well were treated with 10 μM WFA or 30 ng/ml PMA for 12 h in white 96 well flat bottom polystyrene plates . Promoter activity was assessed by the Dual-Glo® Luciferase assay system . Luminescence was measured using the GloMax® Explorer luminometer (Promega). Media background luminescence was subtracted and the ratio of firefly to renilla luminescence was calculated.TK-renilla luciferase vector was a generous gift from the laboratory of Dr. Martha Peterson, Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky, USA. 2.5 × 10post hoc pairwise multiple comparisons as appropriate and p values < 0.05 were considered significant. GraphPad Prism 6.05 was used for statistical analyses .Statistically significant differences between groups were evaluated by Student's t test or Tukey's test for Additional methods are provided in Supplementary Materials.


Human T cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that causes adult T cell leukemia (ATL) in susceptible individuals. The HTLV-1-encoded oncoprotein Tax induces persistent activation of the nuclear factor-κB (NF-κB) pathway. Early growth response protein 1 (EGR1) is overexpressed in HTLV-1-infected T cell lines and ATL cells. Here, we showed that both Tax expression and HTLV-1 infection promoted EGR1 overexpression. Loss of the NF-κB binding site in the EGR1 promotor or inhibition of NF-κB activation reduced Tax-induced EGR1 upregulation. Tax mutants unable to activate NF-κB induced only slight EGR1 upregulation as compared with wild-type Tax, confirming NF-κB pathway involvement in EGR1 regulation. Tax also directly interacted with the EGR1 protein and increased endogenous EGR1 stability. Elevated EGR1 in turn promoted p65 nuclear translocation and increased NF-κB activation. These results demonstrate a positive feedback loop between EGR1 expression and NF-κB activation in HTLV-1-infected and Tax-expressing cells. Both NF-κB activation and Tax-induced EGR1 stability upregulated EGR1, which in turn enhanced constitutive NF-κB activation and facilitated ATL progression in HTLV-1-infected cells. These findings suggest EGR1 may be an effective anti-ATL therapeutic target. HTLV-1 (HTLV-1) . NF-κB a(HTLV-1) . Tax-med(HTLV-1) .EGR1 is an immediate-early response protein that can be rapidly and transiently induced by various stimuli, including growth factors, cytokines, mechanical injury, or shear stress . A subgrin vitro and regulates NF-κB transcriptional activity in vivo [NF-κB p65 subunit-induced transcription of human immunodeficiency virus 1 (HIV) long terminal repeats was previously shown to be dependent on an interaction with the Sp1 zinc finger DNA-binding domain . EGR1, w in vivo , 24. HowOur previous study revealed that Tax induces EGR1 expression . The preOur previous study established a Tax-expressing Jurkat cell line (TaxP) and a Tax-negative control line, TaxN, and demonstrated enhanced EGR1 expression in TaxP cells using microarray analysis , 25. To To determine the elements involved in HTLV-1-mediated EGR1 regulation, luciferase reporter plasmids containing different length segments of the EGR1 promoter region  were constructed and transfected into Tax- or HTLV-1-positive cells Figure . 550 bp The promoting roles of binding sites located in the -413–-85 EGR1 promotor have been reported previously , but theTo further confirm the effect of NF-κB activation on EGR1 expression, we assayed protein levels in p65 shRNA-transfected or Bay 11-7082-treated TaxP and MT2 cells. EGR1 was reduced in both cell lines Figure . Tax mutConsidering that Tax can affect the functions of various transcriptional factors by binding to them, we investigated whether Tax directly interacted with EGR1. To detect interactions between Tax or Tax mutants and ERG1 using the ERG1 antibody, 293T cells were transfected with Tax- or Tax mutants-expressing plasmids and pcDNA3.0-ERG1 for 48 h. Immunoprecipitation assay results showed that Tax, but not its mutants, co-precipitated with ERG1 Figure , indicatWe examined whether EGR1 affected NF-κB activation. TaxP and MT2 cells were transfected with EGR1-expressing plasmids, and EGR1 upregulation led to enhanced NF-κB signaling Figure  and dosePersistent activation of the NF-κB pathway induced by HTLV-1 Tax is the main cause of T cell transformation and ATL , 28, andEGR1 is a nuclear phosphoprotein that was identified because of its early induction following stimulation of mitogens and differentiation factors , 31. EGRThe EGR1 promoter contains several SREs, an AP-1 binding site, two CREs and an Sp1 consensus sequence –39. An aet al. identified the CArG boxes as Tax-responsive cis-acting elements for regulating the cellular immediate early genes, c-fos, egr-1, and egr-2 [Fujii, nd egr-2 . This mind egr-2 . In our in vitro and regulates NF-κB transcriptional activity in vivo [NF-κB and CREB proteins play central roles in HTLV-1-infected T lymphocyte activation . Our res in vivo , 45. The in vivo , 47, indIn summary, our results demonstrate a positive feedback loop between EGR1 expression and NF-κB activation in HTLV-1-infected and Tax-expressing cells. Elevated EGR1 likely contributes to ATL progression by upregulating pro-transformation genes and facilitating constitutive NF-κB activation. Since both EGR1 and the NF-κB pathway play crucial roles in T cell transformation and ATL progression, ERG1 blockade in HTLV-1-infected patients may delay or prevent development of ATL.T cell lines (Jurkat and MOLT4), HTLV-1-infected T cell lines (MT2 and MT4), Hela cells, and 293T cells were cultured at 37°C in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 units/ml), and streptomycin (100 mg/ml). TaxN and TaxP cells were cultured in complete medium supplemented with the antibiotic, G418 .Anti-EGR1 (22008-1-AP) rabbit polyclonal antibody (pAb), anti-Lamin B (66095-1-Ig), and anti-tubulin (66031-1-Ig) were purchased from ProteinTech . Antibodies against IκBα (4814S), p-IκBα (Ser32) (2859S), and p-p65 (8242S) were purchased from Cell Signaling Technology . Anti-p65 rabbit pAb (ab7970) and anti-Tax mouse monoclonal antibody  were bought from Abcam  and Santa Cruz Biotechnology , respectively. The anti-β-actin mouse mAb (60008-1-Ig), as well as the following immunoglobulin G (IgG) reagents: horseradish peroxidase-linked goat anti-mouse (00001-1) and goat anti-rabbit (00001-2), were purchased from ProteinTech. The NF-κB inhibitor, Bay 11-7082 (B5556), and phytohemagglutinin  were purchased from Sigma . 5-(and-6)-Carboxy fluorescein diacetate, succinimidyl ester (CFSE) (C1157), EGR1 siRNA (1299001), and Lipofectamine 2000 (11668-019) were purchased from Invitrogen . Cycloheximide  was purchased from Solarbio .® qRCR Master Mix  according to the manufacturer's instructions. The relative amount of each gene compared to the ACTB internal control, and the fold stimulation were calculated using the 2–ΔΔCT method. EGR1 was amplified using specific primers (sense: CCCCGACTACCTGTTTCCAC and anti-sense: TGGGTTTGATGAGCTGGGAC). Results are the average of three separate experiments.RNA was extracted from cells using the Trizol reagent  and RNeasy mini kit . mRNA was quantified via real-time RT-PCR using the GoTaqTotal protein was extracted from cells using RIPA buffer and separated in 12% SDS-PAGE gel. Proteins were transferred to PVDF membranes  and incubated with primary antibodies followed by anti-rabbit or anti-mouse IgG secondary antibodies conjugated to horseradish peroxidase. Bands were visualized and imaged using enhanced chemiluminescence reagent .The 1397bp DNA fragment of the EGR1 promotor was amplified from genomic DNA using specific primers . This fragment was cloned into the pMD18-T vector (TaKaRa). E1, E2 and E3 fragments were PCR amplified using pMD18-T-EGR1 as a template with the following specific primers: forward E1 5′-GGGGTACCAGCTTCC CCACTTCGGTCCCC-3′, -993–+287 bp; E2 5′-GGGGTA CCGAGGGAGCAACCAGCTGCGA-3′, -443–+287 bp; E3 5′-GGGGTACCGTGCAGGATGGAGGTGCCGG-3′, -341–+287 bp; E4 5′-GGGGTACCGCCATATAAGGAG CAGGAAGG-3′, -414–-85 bp; and reverse 5′-CCCAAG CTTCCTGGACGAGCAGGCTGGAGA-3′  and mutated MutE (mut-NF-κB) constructs were created according to the Quick change protocol  using the following primers: DelE 5′-TTGGAACCAGGGAGGAGGGAGGGAGCGGAAA TGCCATATAAGG AGCAGGA-3′; MutE 5′-GGAGCAA CCAGCTGCGACCCGACTAGTCCATAT AAGGAGCA GGAAGGATC-3′. Total mRNA was extracted from MT2 cells using the RNeasy mini kit. The 1632bp EGR1 complete CDS was amplified via RT-PCR using specific primers . This fragment was treated with HindIII and EcoRI and then cloned into the pcDNA3.0 vector (Invitrogen). pNF-κB-luc plasmids were kindly provided by Haojiang Luan , and pCMV-Bam-Tax and pCMV-Bam plasmids were kindly provided by Shoji Yamaoka . To construct a plasmid expressing full-length Tax with an RFP tag, the Tax CDS was subcloned from pCMV-Bam-Tax into the Bgl II and Hind III sites of the pmCherry-C1 vector. pCMV-M22 Tax and pCMV-M47 Tax plasmids were kindly provided by Edward Harhaj (The University of Miami). p65 shRNA (sc-29410-SH) was purchased from Santa Cruz. EGR1 luciferase reporter plasmid containing EGR1 binding sites was purchased from Yeasen Biotech .Cells were transfected with pNF-κB-luc, EGR1-luc, or different EGR1 promoter -luc plasmids, and enzymatic activity was examined in cell extracts using the Luciferase Assay System  and a 20/20n Luminometer (Turner BioSystems) according to the manufacturer's instructions. All reporter assays were performed in triplicate and repeated at least three times.MT2 or TaxP cells were seeded into 24-well plates and transfected with 50 nM EGR1 siRNA  and/or pNF-κB-luc using Lipofectamine 2000 (Invitrogen). After 48 h, cells were collected for EGR1 detection or for luciferase activity measurement.7) were collected, fixed with 1% formaldehyde for 10 min to crosslink proteins to DNA, and then quenched with 10× glycine for 5 min. After washing with ice-cold PBS, cells were suspended in SDS lysis buffer. Chromatin was then sheared to a manageable size with 5 sets of 10 s pulses on wet ice using a 100-watt model. ChIPs were performed using the chromatin immunoprecipitation Kit . Sheared chromatin was immunoprecipitated using a specific antibody directed against p65, and a nonimmune serum as a negative control. Then, protein/DNA complexes were reversed by incubating samples at 65°C overnight. Associated DNA was eluted and purified according to the manufacturer's instructions. Captured EGR1 regulatory sequences were identified via PCR analysis using the following primers: 5′- CTCCCGGCTTGGAACCAG-3′ and 5′- CC TTCTTCCCTCCTCCCAGA -3′; 179 bp (-476 bp–-298 bp). One microliter of precipitated and purified DNA was subjected to standard PCR, and DNA fragment sizes were analyzed using 2% agarose gel electrophoresis. Detection of specific DNA sequences was performed via qPCR analysis of ChIPed samples using the GoTaq® qRCR Master Mix (Promega). The EGR1 promotor sites bound by p65 protein were determined using qPCR analysis. The CT value at each p65-targeted site was normalized to the negative control, which was set to 1.According to the protocol recommended by Upstate Biotechnology, TaxP cells . An EGR1 monoclonal antibody  was used to capture EGR1 protein and the target protein, Tax, was detected via western blot.t-test or one-way ANOVA. P < 0.05 was regarded as statistically significant.Statistical significance for the luciferase reporter and qRT-PCR assays was determined using the Student's


Scientific Reports 10.1038/s41598-018-22166-3, published online 28 February 2018Correction to: This Article contains an error in Figure 3, in which the confocal microscopy micrograph images are missing. The correct Figure 3 appears below as Figure


Dicer is a key component of RNA interference (RNAi) and well-known for its role in biogenesis of micro (mi)RNA in the cytoplasm. Increasing evidence suggests that mammalian Dicer is also present and active in the nucleus. We have previously shown that phosphorylated human Dicer associates with chromatin in response to DNA damage and processes double-stranded (ds)RNA in the nucleus. However, a recent study by Much et al. investigated endogenously tagged HA-Dicer both in primary mouse embryonic fibroblasts (PMEFs) as well as adult homozygous viable and fertile HA-Dicer mice under physiological conditions and concluded that murine Dicer is exclusively cytoplasmic. The authors challenged several findings, reporting functions of Dicer in mammalian nuclei. We have re-investigated this issue by applying subcellular fractionation, super-resolution microscopy followed by 3D reconstitution, and phospho-Dicer-specific antibodies using the same HA-Dicer PMEF cell line. Our data show that a small fraction of the murine HA-Dicer pool, approximately 5%, localises in the nucleus and is phosphorylated upon DNA damage. We propose that Dicer localisation is dynamic and not exclusively cytoplasmic, particularly in cells exposed to DNA damage. Cytoplasmic Dicer is a key component of the canonical micro (mi)RNA biogenesis pathway. However, a growing body of evidence points toward localisation and activity of mammalian Dicer in the nucleus. A recent study by Much et al., employed an endogenously HA-tagged Dicer knock-in mouse cell line to show that Dicer is exclusively cytoplasmic. This paper challenges several studies reporting various RNA metabolic functions of Dicer in human nuclei. Given the controversy about Dicer’s subcellular localisation, it is essential to address this issue. Employing the same cells as used by Much and colleagues, we combined super-resolution microscopy followed by 3D reconstitution and biochemical assays to show that endogenously tagged HA-Dicer prominently localises in the nucleus under physiological conditions. We demonstrate that DNA damage triggers accumulation of phosphorylated HA-Dicer in the nucleus, confirming previous observations in human cells. Our data indicate evolutionary conservation of nuclear Dicer localisation and function in mammals in response to DNA damage. The endoribonuclease Dicer recognises and processes double-stranded (ds)RNA substrates of various origins into small non-coding (nc)RNA . Dicer aHowever, mechanistic insight in mammalian nuclear Dicer localisation remains largely inconclusive. Analysis of ectopically expressed human Dicer mutants suggest that the dsRNA binding domain (dsRBD) may harbour a cryptic nuclear localisation signal, which is potentially occluded by the helicase domain in the full-length Dicer protein . Indeed,per se ,  x 100%. Intensities of bands were quantified using ImageJ and values for nuclear Dicer were plotted as relative signals normalised to signals from cytoplasmic fractions or non-damaged nuclear fractions, respectively. For example, calculation of amount of nuclear Dicer using 3F10 Ab as shown in 5 cells grown on 6-well multi-well dishes (Corning) were lysed directly in 100 μl 4 x SDS-PAGE sample buffer, 10 μl of lysate was loaded, separated and stained with Ponceau S (Sigma) prior to antibody hybridisation. For semi-quantitative analysis of detection thresholds of HA antibodies, 10 μl lysate of PMEF::HA-Dicer cells, which were grown on 6-well multi-well dishes and lysed in 100 μl 4 x SDS-PAGE sample buffer, was diluted 5 times in a 2-fold serial dilution series. HA signal intensities of 10 μl of either non-diluted sample, i.e. input, or diluted samples, or Ponceau S stainings thereof, were quantified using ImageJ and plotted as relative normalised signals. Signals were plotted, loss of reactivity of HA antibodies was visualised as gap and quantified as delta . For each HA antibody, a Δ was calculated at dilutions steps 1:2 or 1:4 to quantify signals in the near-linear range of sensitivity.Whole cell extracts from approximately 5x 106 wild type PMEF or PMEF::HA-Dicer cells grown on 10 cm dishes (Corning) were trypsinised, washed in cold 1x PBS and centrifuged . Pellets were lysed in 5 volumes WCE lysis buffer  for 20 minutes on ice, sonicated and Benzonase digested as described above. WCE lysates were precleared with protein G agarose beads (Merck Millipore) for 30 min. Samples were incubated with 5 ug primary HA antibodies for 4 hours and pulled down using protein G agarose beads for 45 min. IP samples were washed three times for 10 min with 800 μl WCE lysis buffer , and eluted with SDS-PAGE sample buffer.For immunoprecipitation, approximately 3x 10The following primary antibodies were used: anti-α-Tubulin ; anti-Rad21 ; anti-RNAPII-CTD S2P ; anti-histone H3 ; anti-HA ; anti-HA ; anti-HA ; anti-Lamin C ; anti-KDEL motif ; anti-γH2A.X ; anti-Drosha ; anti-Cyclin E ; anti-Cyclin B1 ; anti-c-Myc ; anti-53BP1 ; anti-pATM/ATR substrates mix ; anti-pChk1 ; anti-pChk1 ; anti-pERK1/2 ; anti-ERK1/2 ; anti-p-p38 ; anti-p21 ; anti-p16 ; and anti-p-DCR-1  .C. elegans epitopes Ser1705/Ser1833. However, human and mouse epitopes differ by one amino acid relative to the original epitope in C. elegans.The p-DCR-1 signals represent a mixture of two individual antibodies, raised against carboxy-terminal murine Dicer epitopes phospho-Ser1712 and phospho-Ser1836 individually in separate rabbits. Murine epitopes Ser1712/Ser1836 are equivalent to human epitopes Ser1728/Ser1852 and 5 wild type PMEFs, Dicer-/- MEFs clone [1A11], and PMEF::HA-Dicer cells grown on 6-well multi-well dishes (Corning) were washed in 1x PBS, fixed on coverslips with 3% Paraformaldehyde in PBS for 10 min, washed and incubated with 50 mM Ammonium chloride in PBS for 10 min, washed in 1x PBS, permeabilised with PBS/0.1% Tween for 5 min and blocked with PBS/10% FBS for 2 hours at 4°C. Primary antibodies were incubated overnight at 4°C in PBS/0.15% FBS. Alexa Flour 488-, 555-, or 647-conjugated secondary antibodies (Invitrogen) were incubated in PBS/0.15% FBS at room temperature for 1.5 hours in a humidified chamber. Cells were washed 3 times for 5 minutes with PBS/0.1% Triton-X 100 between antibody incubations. Nuclei were counterstained and mounted with 6-diamidino-2-phenylindole (DAPI)-containing Mowiol (Merck Millipore).Approximately 3x 10Auto (~6). 3D representation were generated using Imaris 8.4.1 .For confocal imaging, slides were processed on an Olympus microscope, using 60x lens. Samples with 1.5-mm coverslips were imaged using a an FV1000 confocal system on an Olympus IX-81 microscope with photomultiplier tube detectors and Olympus PlanApo N, 60×/1.35NA lens at RT. DAPI-containing Mowiol (EMD Millipore) was used as the imaging medium. DAPI; Alexa Fluor 488, 539, and 635 (Thermo Fisher Scientific) channels were used for acquisition with Olympus Fluoview software. Experimental settings including values of the laser power for each channel, HV, gain and offset parameters were determined at the beginning of each individual imaging session (by assessing background reactivity and saturation levels of each channel) and kept constant over the entire imaging session. ImageJ software (NIH) was used for further processing of the images. All quantifications represent a number of cells that have shown phenotype or % of positive cells, see figure legends for details. n = number of cells. Signals were quantified using RGB profiler . For super-resolution microscopy, cells were imaged at room temperature using an inverted Zeiss 880 microscope fitted with an airy-scan detector. The system was equipped with Plan- Apochromat 63x/1.4 NA oil lens. 488 nm argon and 405nm, and 633 nm diode lasers were used to excite GFP, DAPI and Alexa Fluor 633, respectively. Sequential excitation of each wavelength was switched per line to ensure blue, green and red channels were aligned. Sections of 20 slices with 0.5 μm thick intervals were collected with a zoom value of 600 pixels/μm. Images were processed using Airyscan processing  in 3D with a strength value of 5 murine wild type PMEF or PMEF::HA-Dicer cells were grown to 70–80% confluency on 6-well multi-well dishes (Corning). Approximately 2x 105 wild type human HEK293 cells were seeded on top of the PMEF layer prior to membrane fusion. Mixed cell populations were grown in presence of Cycloheximide (50 μg/ml) for 4 hours prior to fusion. For heterokaryon formation, cells were washed with warm 1x PBS, incubated with 100 μl warm PEG-3000 solution (50% w/v in PBS) for 2 minutes and washed with 1x PBS five times. Heterokaryons were cultured for additional 2 hours in Cycloheximide- containing medium in presence or absence of Etoposide (25 μM) prior to fixation. Alexa Fluor 647-conjugated Phalloidin (Life Technology) was used to stain the cytoskeleton. Preparation of immunofluorescence slides was performed as described in section imaging analysis.Approximately 3x 10S1 FigImmunoblots probing for cyclin E, cyclin B1, c-Myc, as well as total and phosphorylated ERK1/2 kinases, phosphorylated p38 kinase, or cell cycle inhibitors p21 and p16 in PMEF::HA-Dicer whole cells extracts following starvation  or serum stimulation (0.1% FBS +20% FBS). Control, 10% FBS; Rad21, cohesin subunit, loading control; #, unspecific signal.(TIF)Click here for additional data file.S2 Fig-/- knockout MEF cells (clone 1A11) and with (B) serial dilution of PMEF::HA-Dicer whole cell extract. HA signals were quantified as arbitrary units relative to Ponceau S staining and normalised to input . Loss of reactivity of HA antibodies is defined as deltaHA . Values at non-diluted input samples were set to 1. Ponceau S, loading control; #, aberrant signals; Ab, antibody. See also section “(A and B) Immunoblots displaying reactivity of HA antibodies C29F4, HA.11, 3F10 following incubation with (A) whole cell extracts of wild type PMEF, PMEF::HA-Dicer or Dicer(TIF)Click here for additional data file.S3 Fig(A) Confocal imaging of wild type PMEF and PMEF::HA-Dicer co-cultures using HA antibodies C29F4, HA.11 and 3F10 in absence or presence of Leptomycin B (LMB) or Etoposide. Representative merged images are shown.(B) Immunoblots displaying reactivity of HA antibodies C29F4, HA.11 and 3F10 incubated with whole cell extracts of PMEF::HA-Dicer cells following treatment with Leptomycin B (LMB) or Etoposide. #, aberrant signals; Ab, antibody.(TIF)Click here for additional data file.S4 Fig-/- knockout MEFs after treatment with Etoposide. H3, histone 3, loading control; #, unspecific signal. (E) Immunoblots detecting total HA-Dicer  in subcellular fractions of PMEF::HA- Dicer cells in absence or presence of Etoposide. H3, histone H3; CP, cytoplasm; NP, nucleoplasm; CP and NP fractions are loaded in a 1:1 ratio. See also section “wt), PMEF::HA-Dicer (HA) and Dicer-/- MEF (-/-) co-cultures using 3F10 and p-DCR-1 antibodies in absence or presence of Etoposide. Representative merged images are shown.(A) Immunoblots detecting substrates of Ataxia-telangiectasia mutated (ATM) and ATM-related (ATR) kinase activity (pATM/ATR substrates mix antibody), p21 and phosphorylated histone variant H2A.X  levels in PMEF::HA-Dicer whole cell extracts following γ-irradiation  or incubation with Etoposide or Leptomycin B (LMB). (B) Immunoblots displaying reactivity of HA antibody 3F10 and γH2A.X levels after incubation with whole cell extracts  of wild type PMEFs or PMEF::HA-Dicer cells or after immunoprecipitation (IP) using 3F10 antibody in absence or presence of Etoposide. IgG, immunoglobulin heavy chain, loading control. (C) Immunoblot detecting phosphorylated HA-Dicer using p-DCR-1 antibodies following IP with 3F10 antibody as described in (B); #, aberrant signal; M, molecular-weight size marker. (D) Immunoblots displaying reactivity of p-DCR-1 antibodies following incubation with whole cell extracts of wild type PMEFs, PMEF::HA-Dicer cells or Dicer(TIF)Click here for additional data file.S5 Fig(A) Confocal imaging of phosphorylated HA-Dicer (p-DCR-1) and total HA-Dicer (3F10) in PMEF::HA-Dicer cells in presence or absence of Etoposide or after preincubation with Phosphatidylinositol-3-kinase (PI3K) inhibitors. Quantitation indicates cells with shown phenotype in % and number of cell analysed (n) (top panel). RGB profiles of p-DCR-1 (green) and HA-Dicer (red and blue) signals in representative cells. (B) Immunoblots detecting total HA-Dicer  as well as levels of phosphorylated checkpoint kinase 1  and γH2A.X in PMEF::HA-Dicer cells in presence or absence of Etoposide or after preincubation with Phosphatidylinositol-3-kinase (PI3K) inhibitors. Ponceau S, loading control.(TIF)Click here for additional data file.S6 FigTime course confocal imaging of PMEF::HA-Dicer cells stained for phosphorylated Dicer (p-DCR-1), γH2A.X and total HA-Dicer (3F10) following γ-irradiation with a total dose of 10 Gy and recovery for various hours. Quantitation indicates cells with shown phenotype in % and number of cell analysed (n).(TIF)Click here for additional data file.S7 Fig(A) Confocal imaging of wild type PMEF cells stained for phosphorylated Dicer (p-DCR-1), γH2A.X and total HA- Dicer (3F10) following γ-irradiation with a total dose of 10 Gy and 2 hours recovery time.-/- MEFs (clone 1A11). (C) Confocal imaging of PMEF::HA- Dicer cells incubated with Alexa-Flour conjugated secondary antibodies. (D) Confocal imaging of wild type PMEF (wt), PMEF::HA-Dicer (HA) and Dicer-/- MEF (-/-) co-cultures using 3F10 and p-DCR-1 antibodies following γ-irradiation with a total dose of 20 Gy and 2 hours recovery time. Representative merged images are shown. (E) Confocal imaging of PMEF::HA-Dicer cells stained with p-DCR-1, 3F10, and γH2A.X antibodies following γ-irradiation with a total dose of 20 Gy and 0.5 hours recovery time. Quantitations indicate cells with shown phenotype in % and number of cell analysed (n).(B) as in (A), but using Dicer(TIF)Click here for additional data file.S8 Fig-/- knockout MEFs upon γ-irradiation (right); H3, histone 3, loading control; #, unspecific signal. (B and C) RGB profiles (B) and 3D reconstitution of super-resolution microscopy imaging (C) for phosphorylated HA-Dicer  or total HA-Dicer  in non-irradiated (Control) cells or cells irradiated with 10 Gy followed by 0.5–5 hour recovery time. Representative images are shown.(A) Immunoblot detecting phosphorylated HA-Dicer (p-DCR-1) in PMEF::HA-Dicer whole cell extracts in presence or absence of γ-irradiation (left) and immunoblots displaying reactivity of p-DCR-1 antibodies following incubation with whole cell extracts of wild type PMEFs, PMEF::HA-Dicer cells or Dicer(TIF)Click here for additional data file.


In conclusion, our studies show that cell-permeable iron attenuates VEGFR-2 mediated signaling and inhibits tumor angiogenesis.Angiogenesis is important for tumor growth and metastasis. Hypoxia in tumors drives this angiogenic response by stabilizing Hypoxia Inducible Factors (HIF) and target genes like Vascular Endothelial Growth Factor (VEGF). HIF stability is regulated by Prolylhydroxylases (PHD)-mediated modification. Iron is an important cofactor in regulating the enzymatic activity of PHDs. Reducing intracellular iron, for instance, mimics hypoxia and induces a pro-angiogenic response. It is hypothesized that increasing the intracellular iron levels will have an opposite, anti-angiogenic effect. We tested this hypothesis by perturbing iron homeostasis in endothelial cells using a unique form of iron, Ferric Ammonium Citrate (FAC). FAC is a cell-permeable form of iron, which can passively enter into cells bypassing the transferrin receptor mediated uptake of transferrin-bound iron. Our studies show that FAC does not decrease the levels of HIF-1α and HIF-2α in endothelial cells but inhibits the autocrine stimulation of VEGF-Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) system by blocking receptor tyrosine kinase phosphorylation. FAC inhibits VEGF-induced endothelial cell proliferation, migration, tube formation and sprouting. Finally, systemic administration of FAC inhibits VEGF and tumor cell-induced angiogenesis  ReducedEffect of cell-permeable iron on endothelial cell proliferation was determined using Human Umbilical Vein Endothelial Cells immortalized with SV40 antigen (HUVEC-I) and microvascular endothelial cells (MVEC). Initial studies showed a differential sensitivity of HUVEC-I to cell-permeable iron depending on proliferation status. Quiescent endothelial cell viability was not affected by FAC even at a concentration of 35 μM Figure . HoweverNext, we wanted to test whether iron is the active component of FAC responsible for the inhibition of VEGF-A induced endothelial proliferation. For this purpose, an iron chelator, DFX was used. VEGF-A induced endothelial (HUVEC-I) proliferation in the presence or absence of DFX was monitored in real-time. Representative tracings Figure  show thaIn order to investigate the mechanism of inhibition of VEGF-A induced cellular proliferation, we evaluated the effect of cell-permeable iron on cell cycle (HUVEC-I). Cell-permeable iron treatment did not inhibit progression of cell cycle in endothelial cells (HUVEC-I) Figure  and 2B. After establishing the effect of cell-permeable iron on VEGF-A induced endothelial proliferation, we carried out experiments to study the effect of iron on endothelial migration. Endothelial cells (HUVEC-I) were stimulated to migrate by VEGF-A in scratch wound assays. The control endothelial cells efficiently closed the wound by eighty percent after 24 hours in culture. In the presence of cell-permeable iron (17.5 μM and 35 μM), endothelial cell migration was inhibited by 50% and 70% respectively Figure  and 4B. Endothelial cell morphogenesis is an integral part of angiogenesis. Tube-forming assays truly reflect the complex processes of cell migration and rearrangement to establish a network of cells with a lumen. Effect of FAC was then studied on tube formation. There was a concentration dependent effect of iron on endothelial tube formation. While low concentrations of cell-permeable iron (8.8 μM) did not inhibit, higher concentrations (17.5 μM and 35 μM) significantly inhibited endothelial tube formation. In addition to a decrease in the total tube length, there was a reduction in the number of branch points (nodes and junctions) Figure  and 5B. In order to study the mechanism underlying inhibitory effect of iron on VEGF-A induced proliferation and migration, we studied the effect of cell-permeable iron on VEGF-A induced signaling in endothelial cells (HUVEC-I). Cell-permeable iron did not affect the level of constitutive phospho-VEGFR-2 levels. When stimulated by VEGF-A, the phosphorylation of the receptor increased approximately 20-fold (Tyr-1175) and 3.5-fold (Tyr-1214) at 10 minutes. Cell-permeable iron significantly decreased this phosphorylation of VEGFR-2 at Tyr-1175 and Tyr-1214 sites to less than 50% of the control. Cell-permeable iron prevented phosphorylation of the receptor even after 30 minutes of treatment with VEGF-A. Interestingly, total VEGFR-2 levels increased with cell-permeable iron Figure  and 6B. As a consequence of decreased phosphorylation at the receptor level, down stream signaling was affected by iron treatment. Mitogen Associated Protein Kinase  is involved in VEGF-induced proliferation and activation of AKT-pathway increases survival signals in endothelial cells. FAC did not change the constitutive levels of phospho-p44/42 MAPK or phospho AKT. When stimulated by VEGF-A, phosphorylation at both p44/42 MAPK and AKT increased approximately 5-fold and 1.5-fold respectively. FAC, however, caused a significant reduction in VEGF-induced phosphorylation of p44/42 Mitogen-Activated Protein Kinase (MAPK) and the serine/threonine specific protein kinase AKT at 30 minutes and 60 minutes. Cell-permeable iron did not alter the total levels of p44/42 MAPK and AKT Figure  and 6D. Since cell permeable iron not only inhibited VEGF-induced cell proliferation but also migration, we next investigated the status of focal adhesion kinase (FAK), which is a hub of cell migration related signaling machinery. While FAC did not affect constitutive PY397 FAK levels, it decreased constitutive PY861 FAK levels. When stimulated by VEGF-A, phosphorylation increased approximately 1.5 times at both these sites Figure . Iron trin vivo. Geltrex™ plugs containing VEGF-A were implanted subcutaneously in immunocompetent mice. Vascularization of Geltrex plugs was determined after fourteen days. Figure After establishing that cell-permeable iron inhibits VEGF-A-induced signaling in endothelial cells, effects of iron on angiogenesis were determined in vivo.Next, we evaluated the effect of iron therapy on tumor cell-induced recruitment of neo-vasculature in an immunocompetent mouse model using syngeneic tumor cells. Lewis Lung Carcinoma (LLC) cells were reconstituted in Matrigel plugs and subcutaneously implanted into C57/Bl6 mice. Data in Figure Iron and oxygen regulation in a cell are intertwined at many levels . Iron isCell-permeable iron inhibited tyrosine phosphorylation of VEGFR-2 at two critical sites-Tyr-1214 and Tyr-1175. While the phosphorylation of Tyr-1214 recruits NCK/Fyn/PAK-2 complex and activates endothelial cell migration, phosphorylation of Tyr-1175 is important for both endothelial proliferation and migration . Our stuin vivo. Both growth factor-induced and tumor cell-induced angiogenesis were inhibited by iron . Primary HUVEC cultures were purchased from Neuromics . Endothelial cells were cultured in Endothelial cell Growth Media EGM-2™ (Lonza). For growth factor starvation, Endothelial cell Basal Medium-2™ (Lonza) was used. Experiments were performed on primary HUVEC obtained from two different batches and MVEC used in real-time experiments were procured from a single batch. LLC cells were obtained from ATCC  and grown in RPMI 1640 medium supplemented with 10% FBS, 1% glutamine, 1% Penicillin-Streptomycin and 1% sodium pyruvate.Endothelial cells were treated with cell-permeable iron (FAC). FAC has an iron content of 17.5% (16.5–18.5%) and hence iron concentration for experiments was normalized accordingly.® Low-growth factor basement membrane matrix (Life Technologies) was used for in vitro tube formation assays. Matrigel™ basement membrane matrix (BD Biosciences) was used for tumor cell-induced angiogenesis studies. Cytodex 3™ micro carrier beads  were used for fibrin bead assay. Antibodies used were: anti p44/42 MAPK (CST#4696S), anti phospho-p44/42 MAPK (CST#4370S), anti VEGFR-2 (FLK-1) (sc-2651), anti phospho-Tyr 1175 VEGFR-2 (CST#2478S), anti phospho-Tyr 1214 VEGFR2 (ab5475), anti AKT (CST#2920S), anti phospho-Ser 473 AKT (CST#3787S), anti FAK (EMD Millipore 05-537), anti phospho-Tyr 397 FAK (CST#3283S), anti phospho-Tyr 861 FAK (ab4804), anti p38 MAPK (CST#9211S), anti phospho-p38 MAPK (CST#4511S), anti GAPDH (MAB374), anti Beta-Actin (sc-47778), anti PTP1B (sc-1718), anti HIF-2α (sc-13596), anti HIF-1α (NB 100-479), PE conjugated anti-CD31 (BD Pharminogen-52477), FITC-conjugated anti-αSMA (Sigma-F377), FITC-conjugated anti-cleaved PARP  and FITC-conjugated anti-cleaved caspase-3 . Appropriate secondary antibodies conjugated to horseradish peroxidase (Vector labs) or IRDye® secondary antibodies (LI-COR) were used. Fluorescent probes-Hoescht-33342, 4′, 6-diamidino-2-phenylindole (DAPI) and PI were purchased from Molecular Probes (Invitrogen). FAC and DFX were obtained from Sigma. Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies.VEGF-A (Isoform 165) was cloned and expressed in yeast . GeltrexHUVEC-I  were plated in gelatin (0.2%) coated 96 well plates and allowed to attach for 24 hours. Quiescent cultures were treated with different concentrations of iron in EGM-2. All treatments were done in triplicates. Cell viability was measured by using WST-8 (CCK-8) assay. Parallel series of experiments were carried out to determine the effect of cell-permeable iron on VEGF-A induced proliferation of endothelial cells. In these experiments, HUVEC-I cells were seeded and growth factor starved (EBM-2 with 2% serum) for 16 hours. Then cultures were induced to proliferate in the presence of 100 ng/ml VEGF-A in EBM-2 medium containing 2% serum. Cell viability was determined by CCK-8 assay kit. VEGF-A induced proliferation was calculated by subtracting negative control values from all experimental groups. Iron-induced inhibition of endothelial cell proliferation was calculated as a percentage of the positive control (VEGF-A).HUVEC-I  and MVEC  were plated in gelatin (0.2%) coated E-plate 16 (ACEA Biosciences). Growth factor starvation and VEGF-A treatment were done as previously described. Two independent experiments were carried out. Impedance measurement was recorded every 15 minutes for 48–72 hours using the Real-Time Cell Analyzer Dual Plate (RTCA DP) Instrument (ACEA Biosciences).0 cell cycle arrest. After 16 hours of starvation, cells were stimulated to proliferate in EGM-2 complete medium in the presence of 17.5 μM and 35-μM iron for 24 hours and 48 hours. Cells were then fixed in ice-cold 70% ethanol and stained with PI (20 μg/ml) and RNase (200 μg/ml) for 30 min at room temperature. Cells labeled with PI were then analyzed in BD FACS Canto II using FACS Diva software  and 10,000 events were recorded.HUVEC-I cells were plated in gelatin (0.2%) coated 60 mm dishes. Once 75% confluent, they were growth factor starved in EBM-2 with 1% serum overnight to induce GHUVEC-I were growth factor starved and treated with different concentrations of iron in EBM-2 supplemented with 100 ng/ml VEGF-A for 24 and 48 hours, as previously described. At the end of experiment, cells were stained with Hoescht-33342 (12 μg/ml) and PI (2.5 μM) and images were captured at 10× magnification using Leica DMI3000B. Using ImageJ software, cell death was quantified as percentage of PI positive nuclei out of the total Hoescht-33342 positive nuclei.HUVEC-I cells were plated in gelatin (0.2%) coated 60 mm dishes. They were growth factor starved and treated with different concentrations of iron in EBM-2 supplemented with 100 ng/ml VEGF-A for 48 hours, as previously described. Doxorubicin (4 μM) was used as a positive control for induction of apoptotic cell death. Cells were harvested, fixed and stained with FITC-conjugated monoclonal antibodies for cleaved PARP and cleaved caspase-3, as previously described . Cells wHUVEC-I  were plated in gelatin-coated (0.2%) 24 well plates and allowed to attach for 24 hours. Subsequently they were growth factor starved in EBM-2 with 2% serum overnight and a scratch wound assay was carried out, as previously described . The celHUVEC-I cells  were plated in the upper chamber of the CIM Plate-16 (ACEA Biosciences) in the presence of 17.5 μM and 35-μM iron in serum free medium. Migration of cells was monitored in real-time towards a gradient of 100 ng/ml VEGF-A (lower chamber). Impedance measurement was recorded every 15 minutes for 24 hours using the Real-Time Cell Analyzer Dual Plate (RTCA DP) Instrument (ACEA Biosciences).® Low-growth factor basement membrane matrix (250 ul/well) was added to the wells of a 24 well plate. Subsequently, 75,000 live HUVECs were added over the matrix layer and treated with different concentrations of iron in EGM-2 for 16 hours. At 16 hours, images were captured at 4× magnification with the Nikon AZ100M Microscope and the tube length, nodes and junctions were quantitated using the ImageJ Angiogenesis Analyzer plugin.HUVEC-I were plated in gelatin (0.2%) coated 60 mm dishes and pre-treated with different concentrations of iron for 24 hours. GeltrexPrimary HUVEC (between passages 2 and 4) were maintained in EGM-2 media and seeded on Cytodex™3 micro carriers beads for endothelial cell sprout formation as previously described with some modifications . Vessel ® secondary antibody  at room temperature for one hour. Immunoblots were detected with Clarity™ Western ECL Substrate (BIO-RAD) using enhanced chemiluminescence technique and IR fluorescence was detected using Odyssey Imager. Densitometric analyses and quantification of bands were done using ImageJ software.Endothelial cells  were plated in gelatin (0.2%) coated 60 mm dishes. Once 75% confluent, they were growth factor starved in EBM-2 with 2% serum overnight in the absence or presence of 35-μM iron. After 16 hours of starvation, they were stimulated with 100 ng/ml VEGF-A and lysates were collected at 0, 10, 30 and 60 min. Cells were lysed with 100 μL RIPA buffer (Alfa Aesar) containing protease and phosphatase inhibitor cocktail (Thermo Scientific). Protein concentration was measured using BCA™ Protein Assay Kit (Pierce) and 30 μg of protein was loaded. The samples were resolved in SDS-PAGE gels (7.5% and 15%) and transferred to a nitrocellulose membrane (BIO-RAD). Blots were blocked with 5% non-fat milk or 5% BSA in TBST (for phospho-proteins) for one hour at room temperature. Membranes were incubated with primary antibodies in their recommended dilutions overnight at 4°C. Membranes were then incubated with respective anti-mouse, goat or rabbit IgG secondary antibodies (1:3000) conjugated with horseradish peroxidase or anti-rabbit 680 and anti-mouse 800 IRDyen = 7 for both saline and Iron) were surgically removed and snap-frozen in liquid nitrogen. Frozen plugs were sectioned (15 mm), fixed in cold acetone (–20°C) for 10 min, rehydrated and blocked in 1% BSA. Slides were then stained with PE-conjugated anti-mouse CD31 (1:100), FITC-conjugated anti α-SMA (1:500) and counterstained with DAPI. Random areas (5-20) were imaged at 10× magnification using Zeiss Axioplan 2 Fluorescence microscope. CD31-positive vessel length and branch points (nodes) were quantified by morphometric analyses, as previously described [Low-growth factor basement membrane matrix Geltrex™ with added VEGF-A (200 ng/ml) was injected subcutaneously into the dorsal flanks of 6–8 week old female C57BL/6 wild-type mice. The University of Minnesota animal care and use committee approved all animal studies. Geltrex™ was reconstituted with or without 35-μM iron. Next day, one group was treated with 20 mg/kg FAC (in PBS) alternate day intraperitoneally. After 2 weeks of treatment, mice were sacrificed under pressurized CO2 and Geltrex™ plugs  mice were used for tumor cell-induced angiogenesis studies. Lewis Lung Carcinoma (LLC) cells (1 million/ plug) were mixed with Matrigel™ (3:1) and injected subcutaneously  into the dorsal flanks of 6-8 week old female C57BL/6 mice. Matrigel was reconstituted with or without 35-μM iron. When the plugs were visible in all mice (day 4), one group of five mice was treated with 20 mg/kg FAC (in PBS) by intraperitoneal injections on alternate days. Control group of five mice were treated with similar volume of sterile Hanks Balanced Salt Solution following a similar schedule. After 2 weeks of treatment, mice were sacrificed by pressurized CO2 as per approved animal protocol. Matrigel plugs were resected and snap frozen in liquid nitrogen. Frozen Matrigel plugs were then sectioned and processed for CD31 and α-SMA staining, as previously described.Random areas (5-20) were imaged at 4× and 20× magnification using Nikon AZ100M Fluorescence Microscope and Leica DM5500 B microscope. CD31-positive vessel length and branch points (nodes) were quantified by morphometric analyses, as previously described . CD31-po® 6. Differences in mean values between the two groups were analyzed using the two-tailed Student's t-test. P value < 0.05 was considered statistically significant.Data are expressed as means ± SD. Data represent an average of two-four independent experiments. Statistical analyses were performed using GraphPad Prism