BVVL-MNs explained a reduction in axon elongation, partially improved by riboflavin supplementation. RNA sequencing profiles and protein studies of the cytoskeletal structures showed a perturbation in the neurofilament composition in BVVL-MNs. Furthermore, exploring the autophagy–lysosome pathway, we observed a reduced autophagic/mitophagic flux in patient MNs. These features represent emerging pathogenetic mechanisms in BVVL-associated neurodegeneration, partially rescued by riboflavin supplementation. Our data showed that this therapeutic strategy could have some limits in rescuing all of the disease features, suggesting the need to develop complementary novel therapeutic strategies.Riboflavin is essential in numerous cellular oxidation/reduction reactions but is not synthesized by mammalian cells. Riboflavin absorption occurs through the human riboflavin transporters RFVT1 and RFVT3 in the intestine and RFVT2 in the brain. Mutations in these genes are causative for the Brown–Vialetto–Van Laere (BVVL), childhood-onset syndrome characterized by a variety of cranial nerve palsies as well as by spinal cord motor neuron (MN) degeneration. Why mutations in RFVTs result in a neural cell–selective disorder is unclear. As a novel tool to gain insights into the pathomechanisms underlying the disease, we generated MNs from induced pluripotent stem cells (iPSCs) derived from BVVL patients as an  Riboflavin  is a water-soluble group B vitamin and the precursor of the coenzymes flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN)347SLC52A2  and SLC52A3  are responsible for the neurodegenerative disorder previously identified as Brown–Vialetto–Van Laere (BVVL) or Fazio Londe (FL) syndrome910111213RFVT1 does not seem to be associated with a human disease , as no patients with this deficiency have been described.In 2010, it was established that autosomal recessive mutations in the riboflavin transporter genes BVVL syndrome is a rare neurodegenerative disease characterized by progressive ponto-bulbar palsy, bilateral sensorineural hearing loss, muscle weakness, and amyotrophy due to motor neuron (MN) degeneration13149101112161718in vitro patient-specific models allowed the identification of MN pathological features. Patient MNs exhibited a different transcriptional signature with respect to wild-type (WT) MNs (WT-MNs), as demonstrated by RNA sequencing analysis, suggesting impairment in cytoskeletal structure, axonogenesis, and neurotrophic factor metabolism. Indeed, we demonstrated a perturbation in mitochondrial function and in the autophagic pathway. These features represent emerging pathogenetic mechanisms in BVVL-associated neurodegeneration and possible targets for therapeutic intervention, in addition to riboflavin supplementation.In this study, we thus investigated several pathophysiological aspects in induced pluripotent (iPSC)-derived MNs from two BVVL patients (BVVL-MNs). Establishing RFVT2 and the c.1555C > T/c.1255G > A mutations in the RFVT3 gene, respectivelyin vitro pluripotency. The cells expressed pluripotency markers such as NANOG and SSEA-3, as demonstrated by immunocytochemistry analysis  with a multistage differentiation protocol previously developed for human pluripotent stem cellsBecause MNs are selectively affected in riboflavin deficiency pathogenesis, we evaluated whether our BVVL iPSCs could be lineage-committed toward the MN fate in order to define the effect of After 5 weeks under differentiation conditions, MNs were developed that expressed the pan-neuronal marker TUJ1 and the MN-specific transcription factor ISL1. Most of these TUJ1/ISLET1-positive neurons expressed choline acetyltransferase (ChAT) and were positive for the MN marker SMI32, indicating MN maturity . To evalin vitro differentiation protocol generated a high proportion of MNs but also a mixed cell population containing non-MN cells. To further select MNs, we employed a physical strategy based on gradient centrifugation. This selection step allowed the enrichment of cells that were double-positive for ChAT and SMI32 . Less than 1% of iPSC-derived cells were positive for the astrocyte marker glial fibrillary acidic protein.The To reveal disease phenotypes associated with BVVL-MNs, we measured basal MN survival by counting SMI32-positive and ChAT-positive cells. No significant survival reduction was detected in either culture condition .To investigate whether RFVT mutations impair axon stability and elongation, we performed morphological analyses. After 8 weeks of culture, BVVL-MNs exhibited a significantly reduced axon elongation that was improved but partially rescued by riboflavin supplementation, in particular in the patient with mutant RFVT3 . OverallTo identify molecular pathways that are potentially relevant for BVVL pathogenesis, we applied massive parallel RNA sequencing to MNs derived from BVVL patients and controls in the basal condition and after riboflavin treatment.RNA sequencing disclosed 537 differentially expressed transcripts (fold change ≥2 and diverge probability ≥0.8), 246 of which were up-regulated and 291 down-regulated in BVVL cells . As a meTranscripts implicated in ‘cytoskeleton organization’, ‘transcriptional regulation’, and ‘protein translation’ as well as genes implicated in ‘mitochondrial function’ were the most dysregulated terms.in vitro model. One of the most represented gene sets is involved in neurite outgrowth and axon guidance and includes alteration of neurofilament (NF) mRNA, in particular in the NF heavy subunit (NFH), relevant constituents of the cytoskeletal structure, and molecules of the ephrin (EPHA) pathway that are important for axon guidance. Because NF imbalance has been associated with MN diseases24NPTX2), Palladin (PALLD), and Ephrin-A2 (EPHA2), as well as the up-regulation of SLC8A2 in BVVL-MNs in comparison with WT cells. Interestingly, all of these genes are related to neurite outgrowth and guidance. Given the potential relevance of EPHA2 in BVVL axonal defects, we also evaluated the protein level of EPHA2, confirming its downregulation in BVVL cells, without significant modifications after riboflavin treatment  revealed 765 differentially expressed transcripts . RemarkaTo determine the most significant effects of riboflavin treatment in terms of transcriptional changes, we identified the top 30 transcripts most increased and decreased in abundance at a FDR of 5% . GSEA idGene sets that were modified after treatment in BVVL-MNs included terms related to protein and RNA metabolism, mitochondrial function, and autophagy . In partQuantitative RT-PCR was used to validate the expression of some differentially expressed genes related to axon guidance, neuronal metabolism , and proteasome cell function (PSMB8), confirming that their expression pattern shifted toward the normal WT profile after riboflavin treatment . Overallin vitro and in vivo models results in phenotypes closely resembling human MN pathologies2524RNA-seq analysis showed that a significant proportion of all transcripts involved in cytoskeletal and axon guidance were altered in BVVL-MNs . PerturbOur RNA-seq data showed that the NFH was specifically up-regulated in our BVVL-MNs , as confFurthermore, the NF immunofluorescent signal in MNs revealed that the expression levels of NFH were altered . In partTo define whether the transcriptional changes in mitochondrial genes identified by RNA-seq in BVVL-MNs  and 3 weFirst, we assessed for the presence of an OXPHOS or fatty acid β-oxidation impairment in BVVL-MNs, as observed in MADD. Mitochondrial respiration and β-oxidation were not different in BVVL-MNs (with or without riboflavin treatment) with respect to control cells .To then analyze whether a dysregulation of the fusion/fission mitochondrial events takes place in the BVVL-MNs, as occurs in MADD, we examined the expression level of mitofusin2 (MFN2), one of the key regulators of mitochondria fusion degraded during inhibition of fusionWe afterward investigated whether the fusion inhibition events in mitochondria correlate with changes in autophagy. To check for the induction of autophagy, we compared the expression of the autophagy markers LC3B and p62/SQSTM in BVVL-MNs and WT-MNs. A reduction in the LC3B-II/LC3B-I ratio and increase in autophagic substrate p62 demonstrated reduced autophagy in BVVL-MNs with respect to WT-MNs . After rTaken together, our data suggest that a reduction of fusion and mitophagy was present in BVVL-MNs, supporting their limited capacity for renewing mitochondria. Riboflavin exposure normalized the mitophagy rate. These findings showed that pathogenetic pathways in BVVL involve mitochondria turnover but not their functionality. Overall, mitochondrial turnover could represent key pathogenetic components and potential therapeutic targets of BVVL.RFVT2/3 genes; however, the mechanisms by which mutant RFVT proteins induce MN degeneration are largely unknownBVVL and FL are rare genetic MN diseases caused by mutations in in vitro disease modelling and therapeutic discovery in neurodegenerative diseases that involve a specific neuronal populationin vitro BVVL disease model using human patient–specific iPSC-derived MNs. Because surface specific MN markers have not yet been established, we isolated MNs with gradient centrifugation, confirming the generation of pure MNs by immunocytochemical and molecular marker expression.Patient-specific iPSCs represents a novel tool for This system allowed analysis of the impact of RFVT2/3 variants on MN phenotype and to study the effects of riboflavin supplementation. Our studies demonstrated that the BVVL-MNs showed a significant reduction in their axonal length, improved by riboflavin treatment. This phenotype is similar to that observed in MN disease such as SMA203020Considering the cytoskeletal-dependent neuronal pathology observed in BVVL in a broader context, we can group BVVL with those pathologies in which cytoskeletal affect neuropathological features: these defects could be responsible for the neurological motor signs of BVVL. In general, the cytoskeleton is important in a broad series of events regulating neurogenesis and maintenance of neuronal function; therefore, alterations in genes controlling cytoskeletal dynamics can easily lead to severe neurologic diseases3436The improvement in RNA sequencing technologies has allowed identification of specific transcriptional changes in human MN diseases such ALS and SMA, contributing to defining possible disease hallmarks and pathways associated with differential MN vulnerability, thus presenting a number of striking targets for future therapeutic strategy38NPTX2), palladin (PALLD), and EPH receptor A2 (EPHA2), all genes related to neurite outgrowth and axon guidance. NPTX and their corresponding receptors have been studied as synapse-associated proteins in the nervous system and are relevant in promoting neuronal growth and circuitry organization. NPTX2 downregulation can negatively affect these eventsIn this study, for the first time, by applying RNA sequencing analysis to our model in the basal condition and after riboflavin treatment, we identified specific BVVL gene expression changes in RNA/protein metabolism, axonal and cytoskeletal structure, and mitochondrial/oxidative function. Novel candidate genes, with significantly altered expression in BVVL-MNs compared to WT-MNs were selected. In particular, we detected the significant downregulation of neuronal pentraxin 2  and INA , as well as Neurocan (NCAN), a proteoglycan that modulates axonal outgrowth. DNAH10 is a microtubule-associated protein and key component of MN axonsFurthermore, our data showed an alteration in the delicate NF stoichiometric balance, a key pathogenetic event that has been previously suggested to be central in several MN diseases including ALS24495024525325555657In agreement with this observation in ALS-MN, our BVVL-MNs presented an increased level of NFH with aggregation that is likely responsible for neurite degeneration. The substantially higher NF content in MNs demands tight regulation; thus, its deregulation can account for the selective MN degeneration present in BVVL. This finding confirms the potential of targeting NF regulation for therapeutic intervention in MN diseases, but also in BVVL.As noted, riboflavin is the precursor of the active coenzymes FMN and FAD that are essential cofactors for the electron transport chain and the lipid metabolism596163in vitro context, but the physiological environment obviously is 3D and characterized by a tight communication among different cell types . Moreover, the in vitro system allows direct access to the cell of interest whereas in the human body, similar access may be much more challenging. Despite these limitations of our approach, we still consider it to be a useful model for unraveling new aspects of human pathologies, especially in cases where tissue accessibility is hard to achieve. We aim to continue our investigations of BVVL pathogenesis using iPSC-derived MNs in 3D culture systems and co-culturing of glial cells and neurons.In conclusion, iPSC patient-derived MNs allowed us to unravel novel molecular and cellular pathogenetic events that represent possible targets for therapeutic intervention, in addition to riboflavin supplementation for BVVL and other MN diseases. The strategy of using iPSCs obtained from patients to differentiate MNs offers the possibility to study the cell type of interest and to dissect the cellular  and molecular properties of the affected cells. Moreover, this approach allowed us to perform detailed analyses following riboflavin supplementation. Despite these advantages, limitations in understanding the pathogenesis of MN diseases remain. We analyzed the MN cultures in the commonly used 2D 2.The studies involving human samples were conducted in compliance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) and with national legislation and institutional guidelines. Human fibroblast cell lines were obtained from Eurobiobank with informed consent . Fibroblasts were generated from dermal biopsies following informed consent as described previouslyMN differentiation was performed as previously described652067The BBVL cells were cultured at two riboflavin concentrations: 5 nmol/l  and 1 μmol/l (identified as the therapeutic condition). The high concentration of riboflavin (1 μmol/l) was chosen because this concentration is above the level of riboflavin observed in plasma from individuals treated with riboflavin77Cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with Triton 0.25%, and then blocked with 10% bovine serum albumin in 1× phosphate-buffered saline and 0.3% Triton X-100 for 1 h at room temperature. We incubated the cells with primary antibodies to NANOG , SSEA-3 , ChAT , SMI32 , and Neurofilament-200 (1:1000 Sigma) overnight, and then with anti-rabbit or anti-mouse or -goat Alexa Fluor 488 or 594  secondary antibody for 1.5 h at room temperature.Phenotypic analysis was performed as previously described6566Total RNA was isolated from pooled cells using the PicoPure RNA Isolation Kit (Arcturus), which allows the recovery of RNA from a small amount of cells, and quantified using Bioanalyzer (Agilent). A TruSeq RNA sample preparation Kit v2 (Illumina) was used to obtain cDNA libraries from total RNA, and the Nextera DNA sample preparation kit (Illumina) was employed to prepare the sequencing library. Libraries were sequenced on an Illumina HiSeq 2000 employing the standard Illumina pipeline software on an Illumina platform (performed at BGI). Two biological replicates for patients and controls, each comprising four pooled samples, were analyzed. Each replicate was analyzed separately and then pooled. Raw data were processed using TopHat version 2.0.9 and Cufflinks to align genome reads, assemble the data into transcripts, and define expression levels. The aligned mRNA reads were used to identify differentially expressed genes.ftp://gseaftp.broadinstitute.org/.For NGS data analysis, we used two bioinformatics tools that query for enriched gene ontology terms: gene-annotation enrichment analysis with DAVIDACTB and referred to the relevant control samples.Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen). Concentrations were measured with a Nanodrop spectrophotometer. Only samples with ratios between 1.8 and 2.0 were further analyzed. A total of 1 μg of total RNA was reverse-transcribed using the Ready-To-Go kit . Reverse-transcribed material (5 ng for each sample) was amplified using the TaqMan Universal PCR Master Mix (Applied Biosystems) and the appropriate primers to evaluate gene expression (probe ID available upon request) in the 7500 Real Time PCR System. The expression levels for all genes were normalized to the average levels of the housekeeping gene The specific activities of individual respiratory-chain complexes and β-oxidation were evaluated in MNs, as previously described71Proteins extracted from MNs were separated by SDS-PAGE. The primary antibodies used in these experiments were anti EphA2 , anti-NFs , MFN2 , DRP1 , p62 , LC3 I/II , PARKIN , and BNIP3  using the manufacturer instructions. The following secondary antibodies were used: polyclonal anti-rabbit  and polyclonal anti-mouse . The nitrocellulose membrane was stripped and re-probed with anti-actin monoclonal antibody as a loading control. Densitometry was performed using ImageJ software.Stats Direct software was used for all statistical analyses. All counting data from immunocytochemical analyses and cell survival were expressed as mean ± SD or SEM. Differences between two means were analyzed using the Student’s t-test (two-tailed), and differences among more than two means were analyzed using one- or two-way ANOVA. When ANOVA revealed significant differences, pair-wise comparisons between means were performed using Tukey’s post-hoc test.How to cite this article: Rizzo, F. et al. Genome-wide RNA-seq of iPSC-derived motor neurons indicates selective cytoskeletal perturbation in Brown–Vialetto disease that is partially rescued by riboflavin. Sci. Rep.7, 46271; doi: 10.1038/srep46271 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In the present study we investigated the role of H2S in ethylene-promoted banana ripening and senescence by the application of ethylene released from 1.0 g·L−1 ethephon solution or H2S with 1 mM sodium hydrosulfide (NaHS) as the donor or in combination. Fumigation with ethylene was found to accelerate banana ripening and H2S treatment effectively alleviated ethylene-induced banana peel yellowing and fruit softening in parallel with decreased activity of polygalacturonase (PG). Ethylene+H2S treatment also delayed the decreases in chlorophyll and total phenolics, and increased the accumulation of flavonoid, whereas decreased the contents of carotenoid, soluble protein in banana peel and reducing sugar in pulp compared with ethylene treatment alone. Besides, ethylene+H2S treatment suppressed the accumulation of superoxide radicals (·O2−), hydrogen peroxide (H2O2) and malondialdehyde (MDA) which accumulated highly in ethylene-treated banana peels. Furthermore H2S enhanced total antioxidant capacity in ethylene-treated banana peels with the 2,2’-azobis(3-ethylbenz-thiazoline-6-sulfonic acid (ABTS) assay. The result of quantitative real-time PCR showed that the combined treatment of ethylene with H2S down-regulated the expression of ethylene synthesis genes MaACS1, MaACS2 and MaACO1 and pectate lyase MaPL compared with ethylene treatment, while the expression of ethylene receptor genes MaETR, MaERS1 and MaERS2 was enhanced in combination treatment compared with ethylene alone. In all, it can be concluded that H2S alleviates banana fruit ripening and senescence by antagonizing the effect of ethylene through reduction of oxidative stress and inhibition of ethylene signaling pathway.Accumulating evidence shows that hydrogen sulfide (H Musa acuminata, AAA group) is one of the most popular fruits in the world with high nutritional and economical values , and 50 μL of sample extracts. Corrections were made for the background absorbance in the presence of 50 U of superoxide dismutase (SOD). The rate of ·O2− production was expressed as μg·g−1 FW (fresh weight) · s−1.Contents of ·Ou et al.  with sli2O2, banana peels (0.50 ± 0.05 g) were ground and extracted in 3 mL cold acetone. The homogenate was centrifuged at 10,000 g at 4°C for 30 min and 0.5 mL of the supernatant fraction was mixed with 1.5 mL of CHCl3 and CCl4  mixture, then 2.5 mL of distilled water was added and the mixture centrifuged at 10,000 g for 1 min and the aqueous phase collected for H2O2 determination. The reaction system included 0.5 mL aqueous phase, 0.5 mL of buffer , and 20 μL (0.5 unit) of catalase as control or inactive catalase protein . After incubation at 37°C for 10 min, 0.5 mL of 200 mM titanium 4-(2-pyridylazo) resorcinol (Ti-PAR) was added to the reaction mixture for further incubation at 45°C for another 20 min. Absorbance at 508 nm was measured and H2O2 content was indicated as μmol·g−1 FW (fresh weight).For determination of Hg for 30 min, and 1.8 mL of the supernatant fraction was mixed with 1.8 mL of 20% TCA containing 0.5% thiobarbituric acid. The mixture was heated at 100°C for 30 min, cooled, and centrifuged at 15,000 g for 10 min. Absorbance was recorded at 532 nm and the value for nonspecific absorption at 600 nm was subtracted. An extinction coefficient of 155 mM−1·cm−1 was used to calculate MDA content and the content of MDA in banana peels was expressed as μmol·g−1 FW (fresh weight).For MDA analysis banana peel samples (0.50 ± 0.05 g) were ground in 3 mL of 0.1% trichloroacetic acid (TCA) and centrifuged at 10,000 g at 4°C and 10 μL sample extract was used for ABTS assays. 7 mM ABTS solution was prepared 16 h in advance, stored in the dark and then adjusted with methanol to an absorbance of 0.7 at 734 nm. Then an aliquot of 10 μL extract was added to 1 mL of ABTS solution, and measurements at 734 nm were registered each minute for five minutes. Radical scavenging capacity was expressed as the inhibition percentage and was calculated using the formula % radical scavenging activity =  × 100 as described in Girennavar et al. [To estimate the total antioxidant capacity in banana peels, the 2,2’-azobis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS) free radical scavenging activity was determined according to the method described by Re et al. . For ABTr et al. .™5 Real-Time PCR System with SYBR Premix Ex Taq  according to the manufacturer's instructions. PCR procedures were as follows: initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. Primers used for quantitative PCR are shown in MaActin was used as the reference gene. Relative quantification was processed using the method of Delta-Ct.Banana peel samples (0.15 g) were homogenized in liquid nitrogen, and total RNA was extracted by TRNzol RNA Reagent kit (Tiangen) according to the manufacturer’s instructions. The total RNA was used for cDNA synthesis using a reverse transcription kit . Total RNA from five treatments is used for first strand cDNA synthesis in a 20 μL reaction volume containing 4 μL 5 × PrimeScript RT Master Mix. Quantitative PCR is performed using a iQStatistical significance was tested by one-way analysis of variance (ANOVA) using IBM SPSS Statistics , and the results were expressed as the means ± SD (standard deviation).2S or ethylene+H2S to study the possible role of H2S in ethylene-induced fruit ripening and senescence. Our preliminary experiment indicated that 1.0 g·L–1 ethylene donor ethephon solution promoted banana ripening effectively. H2S donor NaHS solutions at 0.5 mM induced minor changes in banana peel color, while 1.0 mM of NaHS was found to alleviate the ripening of banana significantly (data not shown). Therefore, 1.0 g·L–1 ethephon solution or 1.0 mM aqueous solution of NaHS or in combination was applied to postharvest banana. As shown in 2S or ethylene+H2S treatments sustained greenness even after 4 days of storage and became yellow after 5 days. The change in banana peel color was evaluated in L* value in banana fruit compare with control, whereas ethylene+H2S significantly attenuated the increase in peel lightness. H2S treatment alone tended to retain the lowest L* value and only minor increase was found during banana storage. The value of a* indicated chromaticity on a green (−) to red (+) axis, and the results showed that dramatic increase in a* value was observed in ethylene-treated banana fruit in comparison with the lower value in control or ethylene+H2S treatment. H2S treatment was found to maintain a* value at a relative stable level, suggesting that H2S treatment effectively retained greenness in banana peels. Ethylene treatment also induced an increase in b* value in banana fruit while ethylene+H2S significantly attenuated the increase  breakdown, which unmasks carotenoid pigments in the plastids . As showorophyll . The conorophyll . On the  content .2S or H2S alone fluctuated during storage and was significantly higher than that of control and ethylene treatment after 1 day of storage  . To undereatment .2O2 content in banana peel showed a consistent increase both in control and ethylene treatment, which increased nearly 1-fold after 5 days of storage. By contrast, H2O2 content in both ethylene+H2S and H2S treatments remained stable during the first 3 days of storage followed by an increase on Day 5, and was significantly lower than that of control and ethylene treatment  as the donor effectively alleviated ethylene-induced banana peel yellowing and fruit softening. The relative stable content of chlorophyll under ethylene+H2S fumigation compared with ethylene alone, as shown in 2S in preventing chlorophyll degradation. Meanwhile the combination treatment decreased the accumulation of carotenoid in banana peel and reducing sugar in pulp compared with ethylene treatment alone. Consistently, Ammawath et al. [2S treated banana pulp compared with ethylene treated ones.Banana fruit after harvest undergo rapid ripening and senescence associated with loss of nutrients, peel browning and softening, resulting in a short shelf life . The redh et al.  also reph et al. , which e2S treatment was found to delay the decrease in total phenolics, and increased the accumulation of flavonoid compared with ethylene alone, highlighting the protective role of H2S in banana storage. Analysis of ROS such as ·O2− and H2O2 revealed that ethylene+H2S treatment suppressed ROS accumulation. Besides, lipid peroxidation, which was evaluated by MDA content, is one of the most widely used indicators of membrane damage. As shown in 2S significantly attenuated the accumulation of MDA in ethylene-treated banana peels during storage. Further, total antioxidant capacity was determined by ABTS method, which indicating that ethylene+H2S induced higher free radical scavenging activity than ethylene alone. These results indicate that H2S could alleviate ethylene-caused postharvest senescence of banana by reducing oxidative damages.Excessive reactive oxygen species (ROS) and oxidative damage are responsible for fruit senescence and deterioration . Plant c2S on ethylene pathway, we assayed the expression patterns of genes that are involved in ethylene biosynthesis  and signal transduction  in banana peels. Ethylene treatment enhanced the expression of MaACS1, MaACS2 and MaACO1, while the enhancement was greatly attenuated in the combined treatment of ethylene with H2S. However, ethylene+H2S enhanced the expression of ethylene receptor genes MaETR, MaERS1 and MaERS2 compared with ethylene alone. Ethylene receptors act as negative regulators which actively repress expression of ethylene responsive genes in the absence of ethylene and are inactivated by ethylene binding [MaETR, MaERS1 and MaERS2 observed in ethylene+H2S treatment might help to inhibit the effect of ethylene. Fruit softening is associated with cell wall degradation by cell wall hydrolases such as polygalacturonase (PG), pectin methyl esterase (PME), pectate lyase (PL) and cellulose [2S attenuated this enhancement. The pectate lyase (PL) showed an increase in transcript level during banana ripening [MaPL was greatly up-regulated in peels after ethylene treatment, while the expression in ethylene+H2S treatment was repressed, all implying that H2S may play an antagonizing role in the pathway of ethylene.Ethylene plays a crucial role in the ripening and senescence of banana. Ethylene is synthesized from methionine via S-adenosylmethionine and 1-aminocyclopropane-1-carboxylic acid (ACC). Two key enzymes in this pathway are ACC synthase (converting S-adenosylmethionine to ACC) and ACC oxidase (converting ACC to ethylene) , which a binding –43. Thusellulose . And ethellulose –46. In tripening . Consist2S could alleviate postharvest senescence of banana and maintain high fruit quality by decreasing ROS accumulation, improving natural antioxidant contents and radical scavenging capacity, and reducing lipid peroxidation, thereby maintaining the stability of the membrane structure. Besides, we provided strong evidence that H2S may play an antagonizing role in the pathway of ethylene by inhibiting ethylene biosynthesis and signaling pathway.In conclusion, our results indicated that H


Tourette syndrome (TS) is a neurological condition first described by Georges Gilles de la Tourette in 1885. TS was largely thought of as a rare and bizarre condition until the 1960s, when the beneficial effects of neuroleptics on tic symptoms led to an exponential increase in neuroscientific research. Today TS is known to be a relatively common condition that is frequently misdiagnosed due to a combination of its variable manifestation and the waxing and waning of tic frequency and severity. Although there has been a paucity of research on TS compared to other movement disorders, in recent years TS has garnered increasing interest and has shown a number of novel and complex sides, about which much is yet to be learnt. The present article discusses where research has taken us thus far and where it is heading in all the major facets of this fascinating condition.


The protein levels of members of the phosphatidylinositol 3-kinase (PI3K)/Akt/glycogen synthase kinase 3β (GSK-3β)/β-catenin signaling pathway were also examined. Results showed that small-magnitude stress significantly upregulated Runx2 and Collagen I and downregulated PPARγ and C/EBPα expression in BMSCs cultured in adipogenic medium, while large-magnitude stress reversed the effect when compared with unloading groups. The PI3K/Akt signaling pathway could be strongly activated by mechanical stimulation; however, large-magnitude stress led to decreased activation of the signaling pathway when compared with small-magnitude stress. Activation of β-catenin with LiCl led to increased expression of Runx2 and Collagen I and reduction of C/EBPα and PPARγ expression in BMSCs. Inhibition of PI3K/Akt signaling partially blocked the expression of β-catenin. Taken together, our results indicate that mechanical stress-regulated osteogenesis and adipogenesis of rat BMSCs are mediated, at least in part, by the PI3K/Akt/GSK-3β/β-catenin signaling pathway.Osteogenesis and adipogenesis of bone marrow mesenchymal stem cells (BMSCs) are regarded as being of great importance in the regulation of bone remodeling. In this study, rat BMSCs were exposed to different levels of cyclic mechanical stress generated by liquid drops and cultured in general medium or adipogenic medium. Markers of osteogenic (Runx2 and Collagen I) and adipogenic (C/EBP Mesenchymal stem cells (MSCs) have multipotent differentiation capabilities and have been confirmed to differentiate into various lineages, including osteoblasts, adipocytes, chondrocytes, myocytes, and fibroblasts , 2. ThusIn bones, osteoblasts and adipocytes are derived from a common progenitor cell, the bone marrow MSCs (BMSCs) . In partIt has been widely accepted that physiological loading is beneficial in maintaining skeletal integrity and increasing bone strength , while oThe commitment of MSCs to different lineages in response to mechanical stress results from different types, magnitudes, and/or frequencies of forces . Intensiβ (GSK-3β)/β-catenin signaling pathway were investigated to illustrate the mechanism. Our present study thus provides a preliminary insight into the mechanical stress-induced changes of osteogenic and adipogenic differentiation in BMSCs and illustrates the role played by the PI3K/Akt/GSK-3β/β-catenin signaling pathway in the process.Over decades of study, it has become more and more clear that the adipogenic differentiation and osteogenic differentiation of MSCs are competing and reciprocal . Our preα, phosphor-Akt (p-Akt), Akt, GSK-3β, β-actin, and the appropriate anti-rabbit horseradish peroxidase- (HRP-) conjugated secondary antibody were purchased from Cell Signaling Technology, USA. Antibodies against Collagen I, PPARγ, β-catenin, CD29, CD34, CD44, and CD45 were purchased from Abcam, UK. Reagent Sources including LY294002 (PI3K/Akt inhibitor), LiCl, dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), indomethacin, and insulin were purchased from Sigma, USA. The enhanced chemiluminescence (ECL) detection substrate was purchased from Thermo Fisher Scientific, USA.Antibodies against Runx2, C/EBP2 at 37°C. After 4 days, nonadherent cells were removed and medium was changed. Cells were passaged as the first passage when they were 90–95% confluent by trypsinization and seeded at a density of approximately 104 cells/cm2 on round glasses  covered with polylysine. BMSCs phenotypes were confirmed by flow cytometry and analysis of cell surface molecules. Briefly, cells at passage one were washed in PBS and incubated with PE-labeled anti-CD29 (1 : 5), anti-CD34 (1 : 100) anti-CD44 (1 : 50), and FITC-labeled anti-CD45 (1 : 50) for 30 minutes. Then the cells were analyzed on a Flow Cytometer (BD Biosciences). Apoptotic cells were excluded from analysis using propidium iodide (PI).Animal study was approved by the animal research committee of PLA Second Military Medical University. Neonatal Sprague-Dawley rats (7 days) were used for the isolation of BMSCs. Bone marrow was isolated aseptically by flushing the femurs and tibias of rats and then suspended in DMEM/F12 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified atmosphere incubator containing 95% air and 5% COμM dexamethasone, 0.5 mM IBMX, 200 μM indomethacin, 10 μM insulin, 10% FBS, and 1% penicillin-streptomycin.According to absence or presence of different levels of stress intervention and different medium cells cultured in, the cells were divided into experimental and control groups, respectively. Stress parameters were set as follows: 0.15 Hz × 8 cm as small-magnitude and 0.6 Hz × 8 cm as large-magnitude for 30 min. The general medium was DMEM/F12 supplemented with 10% FBS and 1% penicillin-streptomycin, while the adipogenic medium consisted of basic high glucose DMEM supplemented with 1.0 Cells on glasses of experimental groups were exposed to cyclic mechanical stress when they were 90–95% confluent. Briefly, thin cover glass plates were placed over the confluent cell layers on the glasses. The cyclic mechanical stress was adjusted by drops of phosphate buffer solution (PBS) to the glass plates. Cells were subjected to different frequency (Hz) (controlled by drops per second) and height (cm) of mechanical stress . ControlμM LY294002 for 1 h followed by mechanical stimulation (0.15 Hz × 8 cm) for 30 min. Cells without stress intervention were used as controls. Then cells were collected and protein expression levels of signaling pathway  were measured by Western blot. For activation of β-catenin, cells were cultured with either 10 mM LiCl or 10 mM sodium chloride (NaCl) (control) in adipogenic medium for 7 days. Then expression levels of adipogenic and osteogenic marker proteins in BMSCs were examined by Western blot.For inhibition of PI3K/Akt signaling, cells were seeded on glasses as described and when they were 90–95% confluent, the medium was changed by DMEM/F12 with or without 10 γ (1 : 1000), Collagen I (1 : 1000), C/EBPα (1 : 1000), p-Akt (1 : 1000), Akt (1 : 1000), GSK-3β (1 : 1000), β-catenin (1 : 1000), and β-actin(1 : 2000) at 4°C overnight. After washing with TBST, blots were then incubated at room temperature for 1 h with HRP-conjugated secondary antibodies diluted to 1 : 5000 in 5% nonfat milk/TBST. Protein bands were visualized using ECL reagents according to the manufacturer's instructions. The intensity values of each phosphorylated kinase were normalized to the corresponding total protein bands. Unless otherwise stated, β-actin was used as an internal control.Total proteins were extracted from cells by using lysis buffer , according to the manufacturer's instructions. Proteins from each sample were separated using 10% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes using an electroblotting apparatus . After blocking in 5% nonfat milk/TBS-Tween 20 (TBST) solution at room temperature for 1 h, membranes were then incubated with primary antibodies specific for Runx2 (1 : 1000), PPARAccumulation of lipid droplets was used to denote adipogenic differentiation of BMSCs. After fixation in 4% paraformaldehyde, cells were rinsed with 60% isopropanol, followed by freshly prepared Oil Red O solution .P < 0.05 was considered statistically significant.All data were presented as means ± standard deviation (SD). Statistical significance was determined using single-factor analysis of variance (ANOVA) followed by post hoc Bonferroni test to compare between groups. BMSCs identification was done by the phenotypic analysis of the cells. Flow cytometric characterization analysis showed that the cells were homogenously positive for CD29 (97.3%) and CD44 (92.9%) and negative for CD34 (3.41%) and CD45 (1.11%) .The adipogenesis of BMSCs was confirmed by Oil Red O staining of the droplets in cells. Significant development of lipid droplets was observed in BMSCs after exposure to adipogenic medium for 7 days compared with control groups. Application of small-magnitude mechanical stress reduced intracellular lipid droplets while large-magnitude mechanical stress led to an increasing of lipid droplets compared with unstressed groups .α, and PPARγ in BMSCs that were cultured for 7 days with or without mechanical stress were determined by Western blot. The results showed that compared with that of control, Runx2 and Collagen I were upregulated (P < 0.05) while C/EBPα and PPARγ were downregulated, respectively (P < 0.05) after BMSCs were cultured in adipogenic medium for 7 days  in BMSCs compared with unstressed groups. However, as the mechanical stress increased, the effect was reversed. Expressions of C/EBPα and PPARγ in BMSCs increased significantly (P < 0.05) which were even higher than that of unstressed groups. Additionally, large-magnitude stress resulted in decreased expressions of Runx2 and Collagen I when compared with small-magnitude stress groups and unstressed groups, respectively (P < 0.05) . In contrast, with the increasing of mechanical forces, levels of p-Akt (P < 0.05) and β-catenin decreased and GSK-3β (P < 0.05) increased, respectively , suggesting that β-catenin could be activated by LiCl. To explore the influence of β-catenin on the differentiation of BMSCs, cells were cultured in adipogenic medium for a 7-day period in presence or absence of LiCl and adipogenic and osteogenic marker proteins in BMSCs were analyzed by Western blot. As shown in P < 0.05), accompanied by reduction of C/EBPα and PPARγ expressions in BMSCs (P < 0.05). In all experiments, NaCl acted as a negative control for LiCl, which had no effect on β-catenin levels or differentiation of BMSCs [Firstly, activation of μM LY294002 significantly blocked the mechanical stress-induced phosphorylation of Akt compared to the stress loaded cells without inhibitor (P < 0.05). Furthermore, inhibition of PI3K/Akt signaling partially blocked the expression of β-catenin by inactivation of GSK-3β (P < 0.05) under mechanical stress. However, LY294002 itself did not necessarily lead to any significant changes of p-Akt, β-catenin, and GSK-3β in the absence of mechanical stimulation /bone morphogenic protein (BMP), MAPKs, wingless-type MMTV integration site (Wnt) signaling, Hedgehogs (Hh), Notch, and RhoA/ROCK pathways [β-catenin acts as a key transcriptional coactivator and transmits extracellular signals to the nucleus to activate target genes [β is active and β-catenin is constitutively phosphorylated by a destruction complex composed of Axin, APC, and GSK-3β, resulting in low cellular levels of β-catenin. Inactivation of GSK-3β induces the accumulation of cytosolic β-catenin and translocation to the nucleus to activate downstream target genes such as Runx2 and PPARγ [β-catenin by inhibition of GSK-3β. Either GSK-3β inactivation or β-catenin activation block the ability of mechanical signals to induce adipogenesis [β-catenin in MSCs results in decreased osteogenic differentiation in vivo and in vitro [β is essential for mechanical inhibition of adipogenesis [β-catenin enhanced osteogenesis, while inhibiting adipogenesis of MSCs, even under adipogenic culture conditions, which is in accordance with their studies.The differentiation of MSCs is an intensely complex process, involving a large number of signaling pathways, cytokines, and transcription factors. Studies in recent years have demonstrated that a number of critical signaling pathways mediate MSCs differentiation, including transforming growth factor-beta (TGF-pathways , 37, 38.et genes . When thnd PPARγ –41. In Mogenesis . It has in vitro . Resultsogenesis . Our resβ and activate β-catenin [β and β-catenin under different levels of mechanical loading were in response to changes in Akt. Akt activation was responsible for the consequent GSK-3β inhibition, as the expression of GSK-3β increased significantly in the presence of PI3K/Akt inhibitor.The PI3K/Akt pathway is reported to be activated by multiple types of mechanical stress , 45, whi-catenin . Our preβ/β-catenin signaling pathway. However, we hypothesized that the effect of changes from the osteogenic to the adipogenic differentiation of BMSCs when mechanical stress increased is partially mediated by the pathway. In fact, decreased activation of β-catenin by large-magnitude mechanical stress could indeed lead to increased adipogenic differentiation relative to osteogenesis, but the phenomenon that large-magnitude mechanical stress induced more adipogenesis and less osteogenesis than that in the control group indicated that there were some other signaling pathways participating in the process. Specifically, mechanical loading leads to activation of the MAPKs, resulting in increased Runx2 and subsequent expression of osteoblast-specific genes [β/BMP signaling and related downstream key factors that may mediate the regulation of stress-induced differentiation of MSCs, which will promote our understanding of the networks of signaling pathways involved in this process and thus make beneficial use of appropriate mechanical stress and avoid the deleterious effects of pathological forces.Taken together, these results suggest that osteogenic and adipogenic differentiation of BMSCs induced by mechanical stress is regulated by the PI3KAkt/GSK-3ic genes , 48. Furic genes , and insic genes . Our fut


Wnt2b, Wnt3, Wnt11, T-cell factor 7 (TCF7), and Frizzled 8 (FZD8) were modified by bFGF stimulation in fibroblasts. Enzyme-linked immunosorbent assay (ELISA) analysis also showed that Wnt pathway was activated under bFGF treatment. Furthermore, treatment of fibroblasts with lithium chloride or IWR-1, an inducer and inhibitor of the Wnt signaling pathway, respectively, promoted and inhibited cell migration. Also, levels of cytosolic glycogen synthase kinase 3 beta phosphorylated at serine9 (pGSK3β Ser9) and nuclear β-catenin were increased upon exposure to bFGF. Molecular and biochemical assays indicated that phosphoinositide 3-kinase (PI3K) signaling activated the GSK3β/β-catenin/Wnt signaling pathway via activation of c-Jun N-terminal kinase (JNK), suggesting that PI3K and JNK act at the upstream of β-catenin. In contrast, knock-down of β-catenin delayed fibroblast cell migration even under bFGF stimulation. RNA sequencing analysis of β-catenin knock-down fibroblasts demonstrated that β-catenin positively regulated the transcription of bFGF and FGF21. Moreover, FGF21 treatment activated AKT and JNK, and accelerated fibroblast migration to a similar extent as bFGF does. In addition, ELISA analysis demonstrated that both of bFGF and FGF21 were auto secretion factor and be regulated by Wnt pathway stimulators. Taken together, our analyses define a feedback regulatory loop between bFGF (FGF21) and Wnt signaling acting through β-catenin in skin fibroblasts.Skin wound healing is a complex process requiring the coordinated behavior of many cell types, especially in the proliferation and migration of fibroblasts. Basic fibroblast growth factor (bFGF) is a member of the FGF family that promotes fibroblast migration, but the underlying molecular mechanism remains elusive. The present RNA sequencing study showed that the expression levels of several canonical Wnt pathway genes, including  Skin serves as a barrier to protect internal tissues from the external environment, playing a crucial role in life sustenance . Skin woWound healing is a complex biological process  that reqWound healing processes are regulated by numerous growth factors, including FGFs . As one The canonical Wnt signaling pathway, also known as the β-catenin pathway or the β-catenin/ TCF pathway , likewisPrevious study reported that FGF and Wnt/β-catenin signaling involved in collective cell migration , additio-catenin resulted in delay of cell migration. Lastly, suppression of β-catenin in fibroblasts led to repression of bFGF and FGF21, which detected with the identical effect on the activation of AKT and JNK, and the acceleration of cell migration.Here, we performed an RNA sequencing analysis to gain insights into the molecular mechanism of bFGF-mediated wound healing, and identified 830 differentially expressed genes in bFGF-treated versus untreated fibroblasts. GO and pathway analyses distinguished a number of significantly altered molecular pathways, especially in the canonical Wnt signaling pathway. Moreover, GSK3β activity was decreased in response to bFGF treatment, while knock-down of βTo date, several studies have drawn attention to the role of FGF21 in modulation glucose homeostasis by binding to FGF receptor 1c isoform in the present of the cofactor β-klotho. Here, we report for the first time the involvement of FGF21 in fibroblast migration.Taken together, our findings provide a new mechanism whereby bFGF (FGF21) and Wnt signaling are tightly connected to maintain fibroblast cell migration through β-catenin.Human foreskin samples were collected from volunteers at the First Affiliated Hospital, Wenzhou Medical University. All volunteers were informed of the purpose and procedures of this study and agreed to offer their tissue specimens with written consent. All protocols for human studies were approved by the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University.The NIH/3T3 mouse embryo fibroblast cell lines were purchased from American Type Cell Collection  and cultured in DMEM, which contained 5.5 mM glucose, 10% FBS and 1% penicillin-streptomycin. Cells passaged 3–15 times were selected for the following experiments.2 flasks. The flasks were placed horizontally for 1 h and then vertically for 3 h in an atmosphere of 5% CO2 at 37°C. The tissues were then cultured in DMEM supplemented with 5.5 mM glucose, 10% FBS, and 1% penicillin-streptomycin, with subsequent changes of the medium every 3 days. Cultured cells were passaged using 0.25% trypsin (Gibco BRL) after cell confluence reached ∼80%. The human fibroblasts were used for various studies described below after 3–6 passages.While for primary human foreskin fibroblasts, fat was removed from all tissue samples, which were cut into 3 mm strips and incubated with 0.05% dispase neutral protease . The tissues in digestion buffer were incubated with Dulbecco’s Modified Eagle’s Medium  supplemented with 10% FBS (Gibco BRL) and 1% penicillin-streptomycin (Gibco BRL) at 4°C overnight. The epidermis was removed from the dermis, and the dermis was finely minced and placed into FBS-coated 25 cm-catenin knock-down NIH3T3 cells for the RNA-Seq experiments. Cell monolayers were rinsed once with ice-cold phosphate-buffered saline, lysed directly in 3.5 cm culture dishes by adding 1 mL of Trizol Reagent  to each dish, and scraped with a cell scraper. Chloroform (0.2 mL) was added to 1 mL of cell lysate, and total RNA was extracted. Total RNA (2 μg) was reverse-transcribed using a GoScript Reverse Transcription Kit  according to the manufacturer’s instructions. Next, qRT-PCR was performed to quantify the expression of selected genes, as previously described  . Our resescribed . The mRN-catenin knock-down NIH3T3 cells were used for the RNA sequencing experiments. RNA sequencing was performed using a kit provided by LC Biotech, Co., Ltd , and data analyses were performed by Novel Bioinformatics, Co., Ltd .Total RNA extracted from untreated or bFGF-treated  human foreskin fibroblasts and βP-value, where the smaller the FDR, the smaller the error in judging the P-value , Biocarta, and Reactome databases. Fisher’s exact test was followed by Benjamini-Hochberg (BH) multiple testing correction to select the most significant pathway(s), and the threshold of significance was defined by the  the FDR .Cell migration was determined using a standard wound healing scratch assay. Cells were seeded into six-well plates and cultured overnight. Confluent cells were cultured in DMEM supplemented with 0.5% FBS for 24 h, and then wounded by creating a 1 mm linear scratch with a sterile pipette tip. Images of the wounded cell monolayers were taken under a Model IX70 Microscope  at 0, 12, and 24 h after wounding. Cell migration into the wounded area was recorded for 24 h using the same microscope equipped with a CoolSNAP HQ CCD Camera  and MetaMorph Software . All experiments were performed in the presence of 5 mg/mL mitomycin-C to inhibit cell proliferation. Concentrations of LiCl (Sigma) and IM-12 (Selleck), inhibitor of GSK3β and promotor of Wnt signaling, and IWR-1 (Sigma) and XAV-939 (Selleck), inhibitors of Wnt signaling, were tested and shown in Supplementary Figure To observe the relationship between Wnt signaling and bFGF activation in cell migration, cells were pretreated with 1.0 μM IWR-1, 1.0 μM bFGF, or 1.0 μM IWR-1 plus 1.0 μM bFGF for 1 h before wounding. 24 h after wounding, the distance from 20 selected cells and the wound leading edge just after wounding was measured using Image J software .9) , anti-GSK3β , anti-pAKT Ser473 , anti-total-AKT , and anti-phospho-stress activated protein kinase (pSAPK)/JNK Thr183/Tyr185 . After washing three times with TBST, blots were incubated with appropriate secondary antibodies  at room temperature for 90 min. Finally, after washing with TBST, the antigen-antibody complexes on the PVDF membranes were visualized using an enhanced chemiluminescence kit . Protein levels were normalized against GAPDH using Image J software, as previously described  to procure whole cell extracts. KeyGen Nuclear-Cytosol Protein Extraction and KeyGen Mitochondria-Cytosol Protein Extraction Kits  were used for protein isolation from the nucleus and the cytosol, respectively . Proteinescribed .PI3-kinase p110α (Cat. no. sc-39128) was purchased from Santa Cruz Biotechnology, Inc. . NIH3T3 cells were seeded 12 h before transfection, with a cell density of 30–50% confluence at the time of transfection. Next, the siRNA (40 nM) was transfected into the cells using Lipofectamine 2000 (Invitrogen) and Opti-MEMI reduced serum medium (Gibco BRL). After 24 h of transfection, cells were processed by semi-quantitative RT-PCR for the detection of PI3K p110α mRNA levels and Western blotting for the detection of p-GSK3β Ser9 levels.The siRNA targeting the mouse -catenin was as follows: 5′-GAACGCAGCAGCAGTTTGT-3′, as described by 5 cells per well in 24-well plates. After overnight culture, various amounts of lentivirus   were added to the wells in the presence of 4 mg/mL Polybrene . The plates were then centrifuged at 2,500 rpm at room temperature for 1 h and returned to the culture incubator. 24 h after infection, the resulting NIH3T3 cell cultures were analyzed for green fluorescence protein expression  by confocal microscopy. Cells treated with 12 μL lentivirus were chosen for the knock-down experiments described below.The siRNA sequence used for the knock-down of βEnzyme-linked immunosorbent assay was performed to detect the levels of FGFs  and Wnt3a  according to the manufacturer’s instructions. Fibroblasts were seeded in 6-well plates and cultured for 24 h. To determine the assay of FGFs secreted by fibroblast, cell culture supernatants were collected after 24, 36, and 48 h, respectively. To determine the effect of β-catenin on FGFs secretion, after 24 h incubation, the media were replaced with fresh media containing 1.0 μM LiCl, 1.0 μM IWR-1, 0.5 μM IM-12 or 0.5 μM XAV-939, and the cells were then cultured for 48 h. To investigate Wnt3a secretion under FGFs treatment, fibroblasts were cultured in the fresh media containing 100 ng/mL bFGF or FGF21. Then, cell culture supernatants were collected to determine FGFs (bFGF and FGF21) and Wnt3a protein levels.Chromatin immunoprecipitation assay was performed by using a ChIP assay kit  according to the manufacturer’s instructions. Cells were cross-linked with 1% formalin. DNA is extracted from immunoprecipitates of β-catenin Ab . For PCR, 2 uL from 30 uL of DNA extraction was used. The primer sequences for qRT-PCR are listed in Supplementary Table t-test.Statistical analysis was performed with GraphPad Prism 5 Software . All data were expressed as mean ± the standard error (SE). Comparisons between two groups were performed using Student’s P < 0.05 and fold change > 1.5)   .Salmonella infection, hepatitis B, and hematopoietic cell lineage) and nine down-regulated clusters . As shown in Figure 1B, Wnt2B, Wnt11, and Wnt3 were down-regulated, whereas Frizzled 8 (FZD8) and T-cell factor 7 (TCF7) were up-regulated in bFGF-treated fibroblasts. Furthermore, the changes in Wnt pathway genes  were verified by qRT-PCR, and the data were consistent with the RNA sequencing results . These findings indicate that bFGF regulates Wnt signaling genes in fibroblasts.The significance of these altered gene expression levels was further assessed from statistical outcomes by testing for association with biological process GO terms. Pathway analysis was used to assess the significance of the differentially expressed gene sets according to KEGG, which demonstrated the following gene clusters that were significantly altered in bFGF-treated fibroblasts: seven up-regulated clusters  infection, Figures 2A,B; Supplementary Figure Figures 2A,B). In addition, this critical effect of Wnt pathway on wound healing was further confirmed by IM-12 and XAV-939, another common used agonist and antagonist of Wnt signaling, respectively .The results from the cell migration assay indicated that Wnt signaling is important for skin wound healing. β-catenin is known as an essential transcription factor in the Wnt pathway that translocates from the cytosol to the nucleus to activate downstream target genes . WesternFigures 2D,F showed that bFGF and LiCl treatment increased the phosphorylation of GSK3β at Ser9 in fibroblasts, but no significant differences were observed between IWR-1-treated and control groups . Contrarily, LiCl treatment did not change pAKT levels , while pGSK3β Ser9 levels were increased . In view of the fact that bFGF induced the neuroblast migration is mediated by modulating FGFR1 signaling via the PI3K P110 alpha isoform, specifically through the phosphorylation of PI3K downstream effectors, AKT and GSK3β    and neut9 were found in human fibroblasts treated with SP600125, a JNK inhibitor . However, JNK phosphorylation levels did not differ with LiCl treatment . Taken together, these results confirmed that bFGF/PI3K/JNK signaling acts at the upstream of GSK3β.JNK is a downstream regulator of PI3K that is reportedly involved in bFGF-regulated fibroblast migration . In next-catenin-specific siRNA was used to repress β-catenin expression , and LiCl or IWR-1 treatment exerted no effect on the β-catenin knock-down cell line , indicating that β-catenin is necessary to promote bFGF-mediated fibroblast migration.To determine the role of β-catenin in fibroblast migration, βpression . Becausepression . Further-catenin knock-down NIH3T3 cells. We found that 411 genes were differentially expressed in the β-catenin knock-down group. Among them, 201 genes were up-regulated, and 210 genes were down-regulated (P < 0.05 and fold change > 2.0) . Interestingly, FGF21 as well as bFGF expression levels were sensitive to β-catenin. According to the expression patterns summarized in Figure 6A, bFGF and FGF21 were reduced significantly as well as Wnt pathway-related gene FZD8, and Wnt3a was increased by β-catenin-specific siRNA.RNA sequencing analysis was performed to examine transcriptome changes in βFGF21, a secreted protein , which iFGF21 in cell migration, wound healing scratch assay and Western blot were performed on NIH3T3 cells with FGF21 treatment. The results showed that FGF21 promoted cell migration of fibroblasts and β-catenin-suppressed NIH3T3 cells , and neutralization of FGF21 in the culture medium by a FGF21 antibody reduced GSK3β Ser9 phosphorylation levels to the same extent as bFGF . The results showed that the LiCl and IM-12 treatment increased secretion of bFGF and FGF21, but decreased by IWR-1 and XAV-939 treatment. Besides, cell migration was reduced by addition of neutralizing antibody of bFGF and FGF21 in the culture medium  , respectFigure 8). The translocation of β-catenin from the cytoplasm into the nucleus can then activate the downstream transcriptional factors, TCF/LEF, promoting the transcription of Wnt target genes such as FZD8 and Wnt3a, and also inducing the expression of bFGF and FGF21.Migration of dermal fibroblasts is crucial for skin wound repair. bFGF is a member of the FGF family of growth factors, and is an efficacious promoter of fibroblast migration. Recent work showed that bFGF activates the PI3K/Rac1/JNK pathway and NFκB-JNK pathway to accelerate cell migration , 2016. HBasic fibroblast growth factor, LiCl, and IM-12 significantly accelerated cell migration. On the other hand, the Wnt inhibitors, IWR-1 and XAV-939, delayed cell migration, and bFGF somewhat attenuated the effects from IWR-1, which is consistent with previous reports . For LiC9. Normally, GSK3β maintains low cytoplasmic expression of β-catenin via phosphorylation. Phosphorylated β-catenin can be ubiquitinated through the F-box-containing protein, beta-transducin repeat containing protein, an ubiquitin E3 ligase, and then degraded by the proteasome  were altered by bFGF stimulation observed by RNA-Seq assay, indicating that bFGF might not only inactivate GSK3β, but also reduce proteasome-mediated degradation of β-catenin , supporting a role for FGF21 in the skin wound healing. In addition, Western blot analysis showed that exogenous FGF21 treatment activated Wnt signaling in fibroblasts to the same extent as exogenous bFGF. These results indicate that acceleration of fibroblast migration through the PI3K/JNK/GSK3β/β-catenin signaling cascade acts in a feedback loop to regulate the expression of both bFGF and FGF21. However, promoter sequence analysis revealed that the putative β-catenin-TCF4 transcription factor complex binding motifs appeared in the promoter of FGF21, but not in bFGF  Wnt signaling plays a role in FGF-mediated fibroblast migration; (2) PI3K/JNK acts at the downstream of FGF signaling to positively regulate GSK3β/β-catenin activity; (3) a new role for FGF21 is identified in fibroblast migration; and (4) β-catenin also acts downstream of FGF signaling, but in turn activates transcription of XW, YX, YZ, CS, and XL proposed and designed experiments and/or interpreted data; YZ, CS, WHC, YS, and LC performed experiments; HW and NS contributed essential reagents and tools; WTC, TW, CN, and JS participated in writing; WTC and JYS contributed essential reagents and tools and gave conceptual advice; ZZ gave conceptual advice and interpreted data; YX contributed essential reagents and tools, gave conceptual advice and critically revised the paper; LJ supervised the project, interpreted data and wrote the paper.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.


Rumex japonicus Houtt. is traditionally used as a medicinal plant to treat patients suffering from skin disease in Korea. However, the beneficial effect of Rumex japonicus Houtt. on hair growth has not been thoroughly examined. Therefore, the present study aims to investigate the hair growth-promoting effect of Rumex japonicus (RJ) Houtt. root extract using human dermal papilla cells (DPCs), HaCaT cells, and C57BL/6 mice model. RJ induced antiapoptotic and proliferative effects on DPCs and HaCaT cells by increasing Bcl-2/Bax ratio and activating cellular proliferation-related proteins, ERK and Akt. RJ also increased β-catenin via the inhibition of GSK-3β. In C57BL/6 mice model, RJ promoted the anagen induction and maintained its period. Immunohistochemistry analysis demonstrated that RJ upregulated Ki-67 and β-catenin expressions, suggesting that the hair growth effect of RJ may be mediated through the reinforcement of hair cell proliferation. These results provided important insights for the possible mechanism of action of RJ and its potential as therapeutic agent to promote hair growth. It continuously cycles through a growth phase (anagen), an involution phase (catagen), and a resting phase (telogen) . Hair lohe scalp . Minoxidhe scalp . Therefoβ-catenin pathway plays an important role in the hair growth cycle. β-catenin in DPC is markedly activated during anagen phase, resulting in initiation of hair follicle formation, stimulating hair growth and differentiation [β-catenin knock-out mice, the catagen and telogen phases induce premature condition and regeneration of hair follicle is the failure [β-catenin is ordinarily phosphorylated in the cytoplasm by glycogen synthase GSK-3b (beta) and kinase-3b, tagged by an ubiquitin and then dissolved by the proteasome. However, Wnt protein binds to receptors of Frizzled family and β-catenin is accumulated in the cytoplasm and translocated into the nucleus, resulting in induction of growth signals for hair follicle progenitors and promoting differentiation of hair follicular keratinocyte [β-catenin pathway. Targeting the β-catenin pathway is an ideal clinical therapy for preventing hair loss and enhancing hair regrowth. Therefore, many researchers have reported chemicals and natural extracts that stimulate hair growth by targeting the β-catenin pathway [The promotion of hair follicle growth is strongly influenced by various hormones, growth factors, and development-related proteins. Among them, Wnt/ntiation –10. In β failure . In the tinocyte , 12. The pathway –15.Rumex japonicus Houtt., a perennial herb plant belonging to the family Polygonaceae, is widely distributed in the East Asia . It has been used for treating heat phlegm, jaundice, constipation, uterine hemorrhage, and skin disease in traditional medicine [ R. japonicus Houtt. contains a large number of anthraquinones, oxanthrones, and flavones and several reports demonstrate antibacterial, anti-inflammatory, and antioxidant effects in skin disease and inhibitory effect of atopic dermatitis [ R. japonicus Houtt. has not been scientifically proven yet. In this study, we investigated for the first time the stimulating effect of dried roots of R. japonicus Houtt. extract (RJ) and its mechanism on hair growth. For this, we investigated the effect of RJ on cultured DPCs, HaCaT cells, and C57BL/6 mice.medicine –20. R. jrmatitis , 18–23. β-catenin, phospho-GSK3β, and β-actin were obtained from Cell Signaling Technology . Minoxidil was purchased from Hyundai Pharmaceuticals . All other reagents used were of the purest grade available.Bovine serum albumin (BSA), Dulbecco's Modified Eagle's Medium (DMEM), Fetal Bovine Serum (FBS), penicillin, streptomycin, and trypsin were purchased from Gibco-BRL . Dimethyl sulfoxide (DMSO) and 3--2,5 diphenyltetrazolium bromide (MTT) were from Sigma-Aldrich Inc. . Antibodies for Bcl-2, Bax, phospho-Akt, phospho-ERK1/2,  R. japonicus Houtt. were provided by Biobeauty Korea Co., Ltd. . The roots were washed 3 times with tap water to remove impurities and dried at room temperature and then stored at 4°C until use. Dried roots were ground into powder with a grinder and extracted 3 times with 95% ethanol at 25°C for 3 days, after which the extract was filtered through the Advantech number 3 filter paper . The filtered liquid was evaporated using rotary vacuum evaporator . The final step was lyophilization under vacuum to dryness.The dried roots ofμg/mL penicillin-streptomycin-amphotericin B solution. They were grown as monolayer cultures and kept at 37°C in a humidified atmosphere with 5% CO2 for growth.Human dermal papilla cells (DPCs) were obtained from Prof. Ohsang Kown in the Department of Dermatology, College of Medicine at Seoul National University. Human keratinocyte (HaCaT) cells were purchased from American Type Culture Collection (ATCC). DPC is obtained from occipital scalp tissue containing more than 100 hair follicles in twelve healthy male volunteers  according to the approval of Seoul National University Hospital Institutional Review Board (IRB number H-1112-096-390). Before starting this experiment the DPC was cultured for three passages. They were maintained in DMEM supplemented with 10% FBS and 100 4 cells/well in 24-well plates and cultured overnight in growth DMEM media. After changing low serum media (1% FBS), the cells were treated with various concentrations of RJ and incubated for 1 and 3 days. MTT solution (5 mg/mL) was then added to each well and incubated for additional 3 h at 37°C. Finally, DMSO was added to solubilize the formazan salt and the amount of formazan generated was determined by measuring optical density (OD) at 540 nm using GENios® microplate spectrophotometer .Cell viability was measured by the MTT assay as previously described . BrieflyμL of RIPA buffer  containing protease inhibitor cocktail . The scraped cells were allowed to lyse for additional 30 min on ice with periodic vortexing. Cell debris was removed by centrifugation  and the resulting supernatant was collected and determined for its protein concentration using a Bio-Rad protein assay reagent . The samples were separated on 10% SDS-polyacrylamide gel and then transferred to PVDF membranes . Western blot was probed with specific primary antibodies overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody  for 1 hr at room temperature. The blots were visualized by using an enhanced chemiluminescence method  and analyzed using ChemiDoc XRS . Densitometry analysis was performed with a Hewlett-Packard scanner and NIH Image software (Image J).Cells plated on microplate dishes were incubated with various concentrations of RJ and then washed with cold PBS. The treated cells were collected by scraping with 300 β-catenin) was added to the cells and incubated overnight at 4°C. The following morning, the slides were washed three times in PBS and incubated with goat anti-rabbit immunoglobulin conjugated with red-fluorescent dye for 1 h at RT. Subsequently, the stained cells were dropped with Vectashield mounting medium containing DAPI to counterstain cell nuclei . The samples were observed with a Leica fluorescence microscope .The DPC and HaCaT cells were grown on cell culture slide  to 40–50% confluence. After incubation for 24 h, the cells were treated with RJ for another 24 h and then washed with PBS three times for 5 min. The cells were fixed with 4% paraformaldehyde for 10 min and blocked with 3% FBS for 1 h at room temperature (RT). Primary antibody (n = 10) were used for studying the hair growing activity of RJ. RJ was dissolved in a vehicle mixture . The animals in group 1 were topically treated with vehicle mixture, animals in groups 2 and 3 were treated with RJ 4 and 8 mg/mL on dorsal skin once a day for 25 days. Animals in group 4 (positive control group) were treated with 5% minoxidil in the same schedule with RJ treatment. Animals were kept in isolation for a certain amount of time and then housed back to separate cages. To assess changes in hair morphology, the mice were anesthetized and photographed at 0, 6, 11, 16, 21, and 26 days. The ratio of hair growth (grown area/shaved area) on the photographs was analyzed using image analysis program NIS-Elements Basic Research Software .This study was performed by the Korea Institute of Toxicology  in accordance with the Institutional Animal Care and Use Committee . 6-week-old C57BL/6 mice were purchased from Samtako Inc.  and adapted for 1 week. The dorsal portion of 7-week-old mice was shaved with an animal clipper, at which mouse hair follicles were synchronized in the telogen stage. Forty animals in 4 randomized groups (μm tissue sections were stained with hematoxylin and eosin (H&E) to confirm cycle of hair follicle. The percentage of hair follicles in each defined cycle stage at day 26 was calculated. The other part of the tissue was frozen in liquid nitrogen for immunohistochemistry.After 26 days, the mice were sacrificed and their shaved skins were collected. The dorsal skin was cut into two parts for hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) uses. The skin was fixed with 10% neutral formalin and embedded in paraffin blocks to obtain longitudinal and transverse section. 5 β-catenin was performed using rabbit monoclonal antibodies. Immunoreactivity was visualized with an avidin-biotin peroxidase reaction . The peroxidase reaction was developed using a 3, 3′-diaminobenzidine tetrahydrochloride . The sections were counterstained with hematoxylin before being mounted.IHC for Ki-67 and The samples were placed on an adhesive carbon tape attached to a metal stub and coated with platinum using a sputtering device. Photomicrographs of hair fiber were obtained by a SEM using JSM-7610F .p < 0.01 was considered to be statistically significant.The results are expressed as mean ± standard deviation (S.D.). One-way analysis of variance (ANOVA) was used to evaluate the significance of difference between two mean values. μM minoxidil (Mi) was used as a positive control. RJ enhanced the proliferation of DPCs by 132.1 and 147.9% at the concentrations of 50 and 100 μg/mL for 3 days compared with the control . Additionally, RJ increased the expressions of β-catenin and p-GSK3β for 12 h in both DPC and HaCaT cells. Treatment with dose-dependent manner of RJ increased the p-Akt and p-ERK1/2 as well as β-catenin and p-GSK3β in DPC and HaCaT cells for 24 h  and Akt signaling pathways. Therefore, the mechanism of proliferative effects by RJ in DPC and HaCaT cells was determined by western blot. As shown in for 24 h . Nuclearth cells . Interes Figures . Similarμg/mL . Any adverse signs  were not observed at the site of application of RJ for total experimental procedure. Furthermore, the histological data indicated that RJ-treated group (8 mg/mL) showed increase in the number and the size of hair follicles, skin thickness, and an earlier induction of the anagen phase, compared with vehicle group. IHC analysis showed that Ki-67 expression was upregulated in RJ 8 mg/mL group compared to that in vehicle group. Collectively, RJ promoted hair growth and anagen induction via proliferation of hair follicle-related cells and the length and size of hair follicle in in vivo study.The DPC are major hair components and secrete a number of growth factors that can modulate the proliferation of follicular epithelium and follicle development . Severaly anagen . The shaTo elucidate the molecular mechanism underlying the ability of RJ to induce hair growth- promoting effect, the protein levels of MAPK kinase family and Akt, which are hair follicle cell proliferation-related molecules, were checked in the DPC and HaCaT cells. Several reports demonstrated that hair growth agents promoted the proliferation and survival of DPC through the activation of ERK1/2 and Akt –34. The β-catenin seems to be critical in hair loss. Several studies have suggested a relationship between β-catenin signaling and alopecia. When androgen treated DPC in androgenic alopecia patients, β-catenin was significantly decreased in the cytoplasm, while GSK-3β was increased [β inhibitor) enhances hair-inducing ability through stabilization of β-catenin and elevated the alkaline phosphatase and insulin-growth factor-1 gene expressions [β-catenin is key regulator of hair follicle growth and cycling phase [β-catenin-dependent genes is observed. Deletion of β-catenin inhibits formation of hair follicle, while constitutively activated β-catenin produces an expansion of hair follicle fate [β-catenin also induces the transition of hair follicles from the telogen phase to anagen phase. Hair cycle regeneration and anagen phase maintenance are β-catenin dependent [β-catenin pathway. Western blot data showed that the expression levels of β-catenin were upregulated by RJ-treated in DPC and HaCaT cells, compared to those in the control or Mi-treated cells. IHC analysis results showed that the expression of β-catenin was increased in RJ-treated cells. Furthermore, in in vivo study, there was a significant increase of β-catenin expression in RJ-treated group (8 mg/mL) compared with vehicle group. On the other hand, it is recently reported that activation of ERK1/2 and Akt is related to β-catenin signaling pathway [β-catenin/TCF complex through inactivation of GSK3β, indicating that phosphorylation of ERK1/2 and/or Akt induces activation of β-catenin signaling pathway. Therefore, the increase of activation of ERK1/2 and Akt by treatment of RJ might help proliferation of hair cells as well as activation of β-catenin, resulting in induction and maintenance of anagen.Recently, among the development-related molecules in hair follicles, it is known that ncreased , 37. BIOressions , 39. In ng phase . During cle fate –43. β-caependent , 11. The pathway , 45. TheIn conclusion, although RJ has less hair growth effect compared with minoxidil, it stimulated hair growth by inducing anagen phase of hair follicles and proliferation of hair cells. We further showed new insights into possible mechanism of action by which RJ may contribute to the pharmacological intervention of alopecia therapy. However, there is no doubt that the limitations of this study still need further investigations and improvements. Additional clinical trials and studies will be necessary to investigate what components in RJ contribute to its efficacy.To observe the morphological changes on the hair surface, the samples were fixed in 1% osmium tetroxide and then dehydrated in an ascending ethanol series (10% to 100%). After drying, the samples were coated with platinum and observed using JSM-7610F .


The β-galactoside α2-6-sialyltransferase 1  is the principal sialyltransferase responsible for the addition of α2-6-sialic acid to the termini N-glycans on cell surface. Although ST6Gal-I in cancer cell resistance to chemotherapeutics agents has been previously reported, the role of ST6Gal-I in clinical drug resistance of hepatocellular carcinoma (HCC) is not fully understood. In this study, we found that knockdown of ST6Gal-I increased the sensitivity of hepatocarcinoma MHCC97-H cells to docetaxel treatment by instigating the process of apoptosis. Silencing ST6Gal-I expression decreased the survival rate of MHCC97-H cells after docetaxel treatment. Importantly, ST6Gal-I silencing resulted in an increasing of phospho-p38, Bax, Bad, cytochrome c and the cleaved caspase-9, 3 and PARP, while a decreasing of the anti-apoptotic protein Bcl-2. In addition, we found that p38 MAPK and caspase-3 inhibitors can reduce the enhanced apoptosis levels of MHCC97-H cells resulted by either ST6Gal-I silencing or docetaxel treatment. Conversely, exogenous expression of ST6Gal-I in hepatocarcinoma Huh7 cells inhibited apoptotic cell death and prevented docetaxel-induced apoptosis by inhibiting p38 MAPK mediated mitochondrial-dependent pathway. Taken together, these results indicate that ST6Gal-I might play a positive role in mediating the survival of human hepatocarcinoma cells and could be a potential target for gene and antitumor drugs therapy. Liver cancer  is the second most frequent cause of death from cancer  in the world . The freThe β-galactoside α2-6-sialyltransferase 1 ST6Gal-I can catalyze the transfer of sialic acid from CMP-sialic acid to the termini Galβ1-4GlcNAc units of N-glycans . ST6Gal-Docetaxel (DTX) is a semisynthetic analogue of taxane, extracted from needles of European yew. It was reported to be involved in the mechanisms inhibiting microtubules disassembly as well as the enhancement of tubulin polymerization , 18. DocIn this study, we analyzed the role of ST6Gal-I in the apoptosis and sensitivity of hepatocarcinoma cells to docetaxel, and explored the possible molecular mechanisms involved in the regulation of tumor cells apoptosis and chemosensitivity. The results showed that ST6Gal-I knockdown increased the sensitivity of hepatocarcinoma MHCC97-H cells to docetaxel, and ST6Gal-I overexpression inhibited the docetaxel-induced apoptosis in Huh7 cells. In addtion, ST6Gal-I silencing resulted in the activation of p38 MAPK signaling pathway, while a decreasing of the anti-apoptotic protein Bcl-2. Conversely, ST6Gal-I overexpression in Huh7 cells inhibited p38 MAPK mediated mitochondrial-dependent pathway. Together, this study indicates that ST6Gal-I could modulates docetaxel sensitivity in human hepatocarcinoma cells via the p38 MAPK/caspase pathway, and might be a potential target for gene and antitumor drugs therapy.P<0.05)  Figure .Two stable knockdown cell lines and control cells were treated with docetaxel to study the possible functions of ST6Gal-I in modulating the apoptosis and chemosensitivity in hepacellular carcinoma cells. Using Annexin V-FITC/PI staining, we found that the apoptosis rates of ST6Gal-I knockdown cells increased statistically as compared to the control cells in the presence or absence of docetaxel Figure . FurtherTo determine whether the enhanced apoptosis in ST6Gal-I knockdown cells combined with docetaxel treatment is reflected at the level of cell survival, we performed a colorimetric cell viability (CCK8) assay. We found that the ST6Gal-I knockdown cells were less resistant to docetaxel than control cells when exposed to different concentrations for 24h and 48h Figure . These rTo investigate the molecular events involved in docetaxel-induced apoptosis, we checked the role of ST6Gal-I knockdown in the activation of the caspase cascade. The expression of cytochrome c and the cleaved caspase-9, 3 and PARP were investigated by Western-blot analysis. As shown in Figure The role of caspases in the apoptotic cascade have been linked to the activation of the p38 MAPK pathway , we therTogether, these findings suggest that ST6Gal-I silencing could enhanced docetaxel-induced apoptosis, which might be attributed to the potentiation of p38 MAPK mediated mitochondrial-dependent pathway.To further confirm the results aboved, we pretreated ST6Gal-I knockdown cells (shST1) and Negative Control-shRNA cells (shNC) with the specific p38 MAPK pathway inhibitor (SB203580) and selective inhibitor of caspase-3 (Ac-DEVD-CHO) before exposure to 0μM or 50μM docetaxel. We found that in the presence or absence of docetaxel, after treatment with SB203580, the protein levels of pp38, proapoptotic members Bax and Bad, cytochrome c, the cleaved caspase-9, 3 and PARP were inhibited, while the expression of Bcl-2 increased significantly. However, addition of Ac-DEVD-CHO only decreased the protein levels of cleaved caspase-3 and PARP, and there are no apparent changes observed in the expression of ST6Gal-I and p38   Figure . By usin) Figure  and 4C.I) Figure . TogetheTo examine whether ST6Gal-I overexpression could affect the p38 MAPK mediated mitochondrial-dependent pathway, we analyzed the expression levels of related proteins in this pathway by Western-blot assays. The results showed that cytochrome c and the cleaved caspase-9, 3 and PARP levels significantly decreased in ST6Gal-I overexpressed cells compared to control cells either alone or in combination with docetaxel treatment Figures  and 5B. Aberrant sialylation have been reported to correlate with the invasion and metastasis of tumor cells , 14. A sMany cellular stress signaling pathways have been involved in the apoptosis machinery and most of them are executed in an orderly manner. Among the different molecular regulations, the caspase cascade represents a key system. Our results have emphasized a regulatory network where ST6Gal-I facilitates the docetaxel-induced apoptosis through a mitochondrial pathway with the release of cytochrome c which in turn activates caspase-9 . ConsequDocetaxel plays a vital role in the regulation of Bcl-2 phosphorylation, which might through the action of kinases in tumor cells . Among tAlthough not fully understood, the roles of α2,6 sialylation in cancer progression may be exerted by affecting the structures of specific sialylated glycoproteins . It has In summary, ST6Gal-I is involved in the apoptosis and survival of hepatocarcinoma cells by the modulation of p38 MAPK mediated mitochondrial-dependent pathway. Therefore, a better understanding of ST6Gal-I regulation and role in the treatment and diagnosis of hepatocarcinoma may lead to new antitumor strategies that will sensitize unresponsive liver cancers to docetaxel-based chemotherapy.Human hepatoma carcinoma cell line MHCC97-H was purchased from Liver Cancer Institute . Established HCC cell line Huh7 was purchased from the Cell Bank of CAS . Cells were maintained in Dulborcco's modified Eagle's medium (DMEM)(Gibco) and supplemented with 10% fetal bovine serum (FBS) (Gibco). All cells were cultured under 5% CO2 at 37°C.Apa I); Reverse, 5′-CTCGAGG CAGTGAATGGTCCGGAAGCC-3′ (Xho I). Short hairpin RNA (shRNA) specific for ST6Gal-I  and Negative Control-shRNA (NC-shRNA) were obtained from GenePharma . The sequences of the short hairpin RNA were as follows, ST6Gal-I-shRNA: 5′-CACCGTACCAGAATCCGGATTATTTCAAGAGAATAATCCG GATTCTGGTACTTTTTTG-3′ and 5′-GATCCAAAAAAGTACCAGAATCCGGATTATTCTCTTGA AATAATCCGGATTCTGGTAC-3; NC-shRNA:5′-CAC CGTTCTCCGAACGTGTCACGTCAAGAGATTACGT GACACGTTCGGAGAATTTTTTG-3′ and 5′-GAT CCAAAAAATTCTCCGAACGTGTCACGTAATCTCTT GACGTGACACGTTCGGAGAAC. Those shRNAs were annealed and ligated into pGPU6 vector to generate shRNA constructs . Cells were transfected with the mixture of ST6Gal-I-shRNA or NC-shRNA or pcDNA3.1/ST6Gal-I or pcDNA3.1 and Lipofectamine2000TM (Invitrogen) according to the manufacturer's instraction. After 48h, the culture medium was replaced with complete medium containing 800 mg/ml of G418 (Sigma-Aldrich) for selecting stably transtected cells. Stable clones were isolated and expanded for further studies.The CDS region of ST6Gal-I (1218 bp) was cloned into the pcDNA3.1 (−) vector to construct pcDNA3.1/ST6Gal-I. The sequences of the primer pairs were as follows: Forward, 5′-GGGCCCATGATTCACACCAACCTGAAG-3′ , and complementary DNA (cDNA) was synthesized using RT-PCR kit (Takara) according to the manufacturer recommendations. The sequences of the primer pairs were as follows: for ST6Gal-I, 5′-AGCCCTTTTACATCCTCAAG-3′ (forward) and 5′-ATGATGATACCAAGCATCCC-3′ (reverse); for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-CACCCTGTTGCTGTAGCCAAATTC-3′ (forward) and 5′-GACATCAAGAAGGTGGTGAAGCAG-3′ (reverse). PCR reactions were incubated at 94°C for 3 min, followed by 30 cycles of 94°C for 30 s, 57°C for 40 s, 70°C for 50 s, and then a final extension at 72°C for 10 min. The amplified products were separated by agarose gel electrophoresis on 2% gel and analyzed by BioImaging systems .Cell lysates containing 30ug of proteins were subjected to 10 % sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). One gel was subjected to Coomassie Brilliant Blue (CBB) staining and another was transferred to nitrocellulose (NC) membrane . Then incubated with Biotin-labeled lectin Sambucus nigra agglutinin  and streptavidin-HRP  for 1h separately at room temperature. The detection was performed using enhanced chemiluminescence (ECL) kit (Advansta). Densitometry of proteins was analyzed with Gel-Pro software.5 cells were resuspended in 100 ul 1× Annexin-V Binding Solution, and then incubated with Annexin V-FITC and PI solutions at room temperature for 15 min in the darkness before the addition of 400 ul 1× Annexin-V Binding Solution. The cells were analyzed by flow cytometry (BD Biosciences).The apoptosis rate within each group of hepatocarcinoma cells were determined by using flow cytometry analysis with an Annexin V-FITC/PI apoptosis detection kit (Dojindo Laboratories). The cells were prepared with certain concentration of docetaxel (Sigma-Aldrich) for 24 h, harvested, washed twice with cold phosphate-buffered saline (PBS) and 1 × 10Formation of condensed chromatin and apoptosis bodies in hepatocarcinoma cells were quantified with 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. The cells were treated with certain concentration of docetaxel for 24 h and fixed with chilled 4% paraformaldehyde at room temperature for 20 min. The treated cells were then washed with cold PBS and stained with DAPI solution for 3 min in the dark. After washing with cold PBS, stained nuclei were imaged by fluorenscence microscope (Olympus Corp). The apoptotic ratio % = (apoptotic cells) / × 100. Approximately 10 different fields of each sample were taken and analyzed within three independent experiments.3 cells/well, were plated in 96-well plates. After an overnight incubation, cells were treated with 0, 0.01, 0.1, 1, 10 and 100μM of docetaxel for 24 h and 48 h. Cell counting kit-8 (CCK-8) solution (Dojindo Laboratories) was added to each well, and the plates were incubated at 37°C for 2 h. The absorbance at 450 nm was measured with a microplate reader (Thermo Fisher Scientific).Hepatocarcinoma cells and its derivative cells, 2 × 10Protein concentrations were measured using BCA kit (Beyotime). Equal amounts of proteins were subjected to 10-15% SDS-PAGE and transferred to NC membrane. Antibodies against ST6Gal-I , p38, pp38, caspase-3 or GAPDH , Bcl-2, Bax, Bad or PARP , ctochrome c or caspase-9  were used as the primary antibodies. The detection was performed using ECL kit. Densitometry of proteins was analyzed with Gel-Pro software.P < 0.05 was considered as statistical significant. All experiments were performed in triplicate.The quantitative data were presented as mean ± SD and experimental differences were analyzed by one-way ANOVA and two-tailed Student's t-test using SPSS 13.0 software. A value of


Wnt/β-catenin signaling is upregulated in triple-negative breast cancer (TNBC) compared to other breast cancer subtypes and normal tissues. Current Wnt/β-catenin inhibitors, such as niclosamide, target the pathway nonspecifically and exhibit poor pharmacokinetics/pharmacodynamics in vivo. Niclosamide targets other pathways, including mTOR, STAT3 and Notch. Novel benzimidazoles have been developed to inhibit Wnt/β-catenin signaling with greater specificity. The compounds SRI33576 and SRI35889 were discovered to produce more cytotoxicity in TNBC cell lines than in noncancerous cells. The agents also downregulated Wnt/β-catenin signaling mediators LRP6, cyclin D1, survivin and nuclear active β-catenin. In addition, SRI33576 did not affect mTOR, STAT3 and Notch signaling in TNBC and noncancerous cells. SRI35889 inhibited mTOR signaling less in noncancerous than in cancerous cells, while not affecting STAT3 and Notch pathways. Compounds SRI32529, SRI35357 and SRI35361 were not selectively cytotoxic against TNBC cell lines compared to MCF10A cells. While SRI32529 inhibited Wnt/β-catenin signaling, the compound also mitigated mTOR, STAT3 and Notch signaling. SRI33576 and SRI35889 were identified as cytotoxic and selective inhibitors of Wnt/β-catenin signaling with therapeutic potential to treat TNBC in vivo. Of all cancers, breast cancer affects women the most with an incidence of about 12%. High morbidity of this disease is associated with triple-negative breast cancer (TNBC), which accounts for 10–20% of all breast cancers  The moniUnderlying signaling characteristics of TNBC may be exploited for developing novel therapies. Wnt/β-catenin signaling drives tumorigenesis in a variety of cancers and contributes to progression of TNBC compared to that of other breast cancer subtypes ,6,7,8. WUnlike colorectal cancers, of which 80–90% contain APC mutations, TNBC generally lacks mutations in Wnt/β-catenin pathway related proteins. Instead, pathway dysregulation is mediated by activation of β-catenin-mediated transcription ,13 and hNovel small molecule inhibitors targeting Wnt/β-catenin signaling in cancers have been recently developed . AppealiMaintenance of cancer stem cells (CSCs), a subpopulation of cells involved in self-renewal and regeneration of tumors, has been associated with Wnt/β-catenin signaling. Breast CSCs, identified by CD44+/CD24− markers, have the capacity to generate tumors ,29. High1/2 = 29 min in rat liver microsomes) and solubility . Ideally, the pharmacokinetic t1/2 should be at least 60 min, requiring twice daily dosing.High-throughput screening of more than 4000 clinically approved compounds revealed the anthelmintic agent niclosamide as a potent Wnt/β-catenin signaling inhibitor with anticancer activity . Current2 group in niclosamide, known to elicit toxic effects, was removed from most of the inhibitors. Since niclosamide was postulated to affect Wnt/β-catenin signaling separately from ATP homeostasis, Mook et al. (Duke University) developed novel compounds to improve Wnt/β-catenin selectivity. The structure of a selective compound evaluated in this study, SRI33576, was first identified by Mook et al. to have specificity for Wnt/β-catenin signaling over ATP homeostasis in colorectal cancer cells +: 296.9992, found: 296.9993.This compound was prepared from 3,5-dichlorobenzene-1,2-diamine and 5-fluoro-2-hydroxybenzaldehyde using Method A. Yield 21%. TLC Rf = 0.45 . 1H NMR  δ 12.60 , 8.54 , 8.22–8.11 , 7.82 , 7.24–7.09 . HRMS m/z calcd. for C13H7ClF2N2O [M + H]+: 280.0215, found: 280.0215.This compound was prepared from 4-chloro-5-fluorobenzene-1,2-diamine and 5-fluoro-2-hydroxybenzaldehyde using Method A. Yield 64%. TLC Rf = 0.30 . 1H NMR  δ 12.99 , 8.24 , 7.95 , 7.70 , 7.45–7.40 , 7.08 . HRMS m/z calcd. for C13H7Cl3N2O [M + H]+: 312.9697, found: 312.9696. HPLC: 100% (tR = 3.05 min).This compound was prepared from 4,5-dichlorobenzene-1,2-diamine and 5-chloro-2-hydroxybenzaldehyde using Method B. Yield 92%. TLC RAll experimental compounds were synthesized at Southern Research  and dissolved in DMSO at stock concentrations of 10 mM. Antibodies purchased from Cell Signaling Technologies  include phospho-LRP6 (#2568), LRP6 (#2560), cyclin D1 (#2978), survivin (#2808), phospho-4EBP1 (#9451), 4EBP1 (#9452), phospho-STAT3 (Tyr705) (#9145), STAT3 (#4904), Hes1 (#11988), α-tubulin (#2144) and lamin A/C (#2032). Tissue culture media were obtained from ThermoFisher  and FBS was obtained from Atlanta Biologicals . Niclosamide, insulin, cholera toxin, hydrocortisone, protease and phosphatase inhibitor cocktails were purchased from Sigma-Aldrich . Epidermal growth factor was obtained from PeproTech .2.Nonmalignant mammary cell line MCF10A and TNBC cell lines MDA-MB-231 and MDA-MB-468 were purchased from ATCC . MCF10A cells were cultured in DMEM/F-12 (1:1) supplemented with 5% horse serum, 10 µg/mL insulin, 0.5 µg/mL hydrocortisone, 0.02 µg/mL epidermal growth factor, 0.1 µg/mL cholera toxin. MDA-MB-231 and MDA-MB-468 cell lines were cultured in DMEM with 10% FBS. SUM149 and SUM159 cell lines were purchased from Asterand  and cultured in DMEM/F12 with 5% FBS, hydrocortisone and insulin. All cell lines were maintained in an incubator at 37 °C and 5% CO3) were seeded with appropriate growth media containing 10% FBS in each well of tissue culture-treated 96-well black plates (Corning #3904)  16 h prior to treatment. Cells were treated with SR compounds for 3 days at concentrations of 0–5 µM. Cell viability was then evaluated with Cell Titer Glo  and luminescence was measured using a TOPCount NXT plate reader . Curves were fit according to nonlinear regression and IC50 values were calculated using GraphPad Prism 7. Cells were treated in quadruplicates and experiments were performed three times.MCF10A and TNBC cells (2 × 10Cells were seeded in 6-well tissue-culture treated plates (Corning) for overnight incubation. Culture media was replaced with growth media containing 1% FBS. Cells were treated with compounds for 48 h prior to harvest. Apoptosis/necrosis was detected by the FITC Annexin V Apoptosis Detection Kit I from Becton Dickinson . Cells positive for Annexin V, propidium iodide (PI) or both were all designated as apoptotic.Cells were seeded in 6-well tissue-culture treated plates for 48 h until 80% confluency prior to treatment. Whole cell extracts were prepared with Laemmli buffer  supplemented with protease and phosphatase inhibitors and equivalent amounts of proteins were separated by SDS-PAGE. Proteins were transferred to polyvinylidene difluoride (PVDF) membrane  and blocked with 5% BSA in tris-buffered saline (TBS) supplemented with 0.1% Tween-20 for 1 h. Membranes were incubated with primary antibody overnight on a shaker at 4 ºC. They were then washed and incubated with secondary antibody for 1 h. Bands were visualized with the Enhanced Chemiluminescence Detection reagents (PerkinElmer).Cells were lysed using the CERI and CERII reagents of the cytoplasmic-nuclear extraction kit (ThermoFisher), to obtain cytoplasmic fractions following treatment. Nuclear fractions were isolated by using 1× Laemmli buffer and sonication for a few seconds. Membrane was incubated overnight with active β-catenin , lamin A/C and tubulin antibodies.Cells were seeded on coverslips for 48 h prior to treatment with DMSO and SR compounds for 18 h. Cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min and permeabilized with 0.5% Triton X-100 for 20 min. Following blocking with 3% BSA for 30 min, cells were incubated overnight with active β-catenin antibody (MilliporeSigma) at 4 °C. Cells were incubated with Alexa 488 goat anti-mouse secondary antibody (1:1000) at room temperature for 1 h. To counterstain, cells were incubated with 1:5000 Hoechst 33342 for 20 min. Coverslips were mounted with Prolong Gold Antifade Reagent (ThermoFisher) and cells were observed with Nikon A1R confocal microscope . Images were obtained at 20× magnification.t test. p < 0.05 values were considered significantly different. Results are shown as mean ± SD.Statistical analyses were performed by the unpaired Student