Musa spp.), which includes dessert and cooking varieties, is found in more than 120 countries and is a member of the order Zingiberales and family Musaceae. The production of bananas requires a certain amount of precipitation throughout the year, and its scarcity reduces productivity in rain-fed banana-growing areas due to drought stress. To increase the tolerance of banana crops to drought stress, it is necessary to explore crop wild relatives (CWRs) of banana. Although molecular genetic pathways involved in drought stress tolerance of cultivated banana have been uncovered and understood with the introduction of high-throughput DNA sequencing technology, next-generation sequencing (NGS) techniques, and numerous “omics” tools, unfortunately, such approaches have not been thoroughly implemented to utilize the huge potential of wild genetic resources of banana. In India, the northeastern region has been reported to have the highest diversity and distribution of Musaceae, with more than 30 taxa, 19 of which are unique to the area, accounting for around 81% of all wild species. As a result, the area is regarded as one of the main locations of origin for the Musaceae family. The understanding of the response of the banana genotypes of northeastern India belonging to different genome groups to water deficit stress at the molecular level will be useful for developing and improving drought tolerance in commercial banana cultivars not only in India but also worldwide. Hence, in the present review, we discuss the studies conducted to observe the effect of drought stress on different banana species. Moreover, the article highlights the tools and techniques that have been used or that can be used for exploring and understanding the molecular basis of differentially regulated genes and their networks in different drought stress-tolerant banana genotypes of northeast India, especially wild types, for unraveling their potential novel traits and genes.The enormous perennial monocotyledonous herb banana ( Musa spp. are seeded and propagated by both sexual and vegetative methods, while the cultivated bananas are sterile and parthenocarpic [Drought is a major threat arising out of unstable environmental conditions and affecting the global agricultural sector and population ,2. It isnocarpic . Though nocarpic . CWRs ofnocarpic , sorghumnocarpic , barley nocarpic , wheat [nocarpic , and alfnocarpic  have beenocarpic . There inocarpic  that neenocarpic ,35.The use of technological advancement in high-throughput phenotyping, transcriptomics, and proteomics helps to identify and understand the genetic basis of drought stress tolerance in the genetic resources of CWRs ,38,39,40The incorporation of stress-tolerant genes, which are naturally present in banana CWRs, is required for producing improved varieties with better performance ,42. Drouhttps://www.fao.org/faostat/en/#data accessed on 22 November 2022). There are over 300 different types of bananas worldwide  are monocotyledonous herbs of the Musaceae family of the Zingiberales order [Musa and Ensete in the family, while others have reported that the family has six genera and 130 species [Musa can be divided into four sub-divisions based on chromosome number: Australimusa, Callimusa, Eumusa, and Rhodochlamys, all of which contain edible cultivars. Therefore, individuals with eleven chromosomes (2n = 22) exhibit the traits of Rhodoclamys and Eumusa. Additionally, the traits of Australimusa and Callimusa can be distinguished while having ten chromosomes (2n = 20). The majority of cultivars are members of the largest and most widespread geological section, known as Eumusa, and are descended from M. acuminata Colla and M. balbisiana Colla) [M. acuminata Colla and M. balbisiana Colla, which yield fruits, are also known by the common name “banana”. Even though there are many different banana varieties, such as the Petite Naine, Poyo, Williams Lacatan, and Grande Naine, the subgroups of M. acuminata known as Cavendish and Plantain are prominent in the global banana market.Bananas  . These tM. acuminata (A) and M. balbisiana (B) by intra- or inter-specific crossing, complex initial cultivation, and dispersal over time, resulting in domesticated parthenocarpic, sterile, polyploid cultivars  [Cultivated bananas are developed from the two ancestors nd ABBB) ,53,54. End ABBB) ,56. Howend ABBB) ,58.In India, the largest area under banana cultivation is in Tamil Nadu, followed by Maharashtra, Gujarat, Andhra Pradesh, Karnataka, and other states following the specific trend of cultivating different banana varieties .https://www.fao.org/faostat/en/#data accessed on 22 November 2022). The preliminary results of FAO-2021 on the banana market review ascertained that Indian banana exports increased significantly from 2018 to 2020 by 233%, or 2.3-fold  and China (13.8%) (2.3-fold A. In the2.3-fold B. The prM. acuminata (A) and M. balbisiana (B) were crossed intra- or inter-specifically to produce domesticated banana cultivars [Wild-seeded ultivars . The banultivars . It alsoultivars . In contultivars ,63. Seveultivars ,64. Wildultivars ,65,66.M acuminata (A genome) had the highest intra-species diversity and was classified into ten subspecies [M acuminata ssp. burmannicoides has been used in various crosses for improved traits as it is tolerant to black Sigatoka and Fusarium wilt race 1 [M. acuminata ssp. macrocarpa, ssp. zebrina, ssp. burmannica, ssp. malaccensis, ssp. burmannicoides, ssp. errans, ssp. truncata, ssp. siamea, and ssp. banksii [M. balbisiana, another important wild progenitor of ‘genome B,’ is also used as a source of stress-tolerant genes, such as Xanthomonas resistance [Musa spp. have also been reported, such as M. haekkinenii [M. itinerans [M. serpentina [M. serpentina species found along the border of Myanmar shows the characteristics of both M. acuminata and M. laterita [M. balbisiana have highly intraspecific diversity, even though there are no variable subgroups reported so far, while ten subgroups have been identified within M. acuminata [M. acuminata subspecies that are the main ancestors of the A genome cultivated bananas: M. acuminata ssp. banksii, M. acuminata ssp. zebrina, and M. acuminata ssp. malaccensis [bspecies . M acumit race 1 . Most of banksii ,67,68. Msistance  and drousistance . Severalkkinenii , M. itintinerans , and M. rpentina . Among tlaterita . The divlaterita . The procuminata . It alsoaccensis . Severalaccensis ,74. The accensis ,76. In aaccensis .M. acuminata subsp. burmannica, M. balbisiana, Musa basjoo, M. itinerans, M nagensium, M. ruiliensis, M. velutina, and M. yunnanensis were found to be tolerant against Foc-TR4 [burmannica is tolerant to Sigatoka leaf spot diseases [M. balbisiana species is composed of a variety of wild types and is a fantastic source of genes that are resistant to a wide range of biotic or abiotic challenges. It is resistant to Fusarium wilt, Sigatoka, Septoria, and Cordana leaf spots, rust, and bacterial diseases, such as head rot (Erwinia spp.), in addition to tolerance for the rhizome weevil (Cosmopolites sordidus Germar) and pseudostem weevil (Odoiporus longicollis) [M. balbisiana, in addition to biotic stresses [ Foc-TR4 , whereasdiseases . Althougicollis) . Abioticstresses .Musa [The genotype of the plant has the potential for stress tolerance; however, water management, good scientific cultivation practices, and crop management can minimize the gap between maximal and actual production . ExploraMusa .Ensete and Musa, in India, and 37 wild taxa have been reported, with the majority of them occurring naturally in northeast India [M. acuminata, burmannicoides, burmannica, and banksii, have been reported in India, occurring in the south and middle Andamans, Western Ghats (Karnataka), Kaziranga Forest (Assam), and Khasi hill ranges  [M. balbisiana is found in northeastern states, including the Andaman and Nicobar Islands, Bihar, West Bengal, Orissa, Kerala, Karnataka, Tamil Nadu, and Andhra Pradesh [M. balbisiana variants have taken on the function of commercial variations [The family Musaceae is represented by the two genera, st India ,84. Figughalaya) . These rghalaya) . However Pradesh . Genes r Pradesh ,86. Due riations .Musa (sect.), seven have been reported in India: M. sikkimensis, M. ingens, M. nagensium, M. cheesmani, M. basjoo, M. itinerans, and M. flaviflora [M. itinerans, a rare species, has been reported from Manipur, Arunachal Pradesh, and Mizoram [M. nagensium is frequently found in the damp evergreen forests of the northeastern states of Meghalaya, Arunachal Pradesh, and Manipur. In Arunachal Pradesh, M. nagensium [Musa aurantiaca is also present [M. nagalandiana, a very rare species of the genus Musa, is named after Nagaland, where it was first identified, and is found growing on hillside slopes in semi-evergreen tropical forests close to the Doyang river in the Zunheboto region of Nagaland [M. puspanjalia in honor of his mother, Puspanjali Gogoi. Large groups of this species are frequently observed on stream banks and hill slopes. Another northeast Indian species, M. kamengensis, is frequently observed on the Jamiri mountain slopes at a height of 1500 m in the West Kameng region of Arunachal Pradesh. It appears to be cold-tolerant [M. aurantiaca specimen was discovered in Assam, and the variety name homenborgohainiana was derived after Homen Borgohain (an Assamese writer) [M. aurantiaca var. jengingensis was given its name after Jenging, the location where it was originally noticed. Quite different from the others, the male inflorescence of this variety vanishes before the fruits reach maturity [M. mannii var. namdangensis of Assam, which took its name from Namdang, the location of its origin, and was only observed in this region. [Among the 37 known species of aviflora ,88. M. i Mizoram . M. nage present . M. nagaNagaland . Rajib Gtolerant . The fir writer) . The varmaturity . The fir region. . The Ind region. ,95.Musa spp. genetic resources naturally in the northeast Indian region, which have remained mostly uncharacterized and untapped, provides vast opportunities for the evaluation of various novel traits, such as stress tolerance during drought. The following is a summary of strategies for the evaluation of drought-induced responses in these different genotypes.The occurrence of a huge reserve of diverse wild Musa spp. found in northeast India , P2O5 (200 mg/L), and N (200 mg/L) should be added to the substrate as nutrients  content 2O2) activity [Hydrogen peroxide (Hactivity Proline content Soluble sugar content Endogenous Abscisic acid (ABA) content Photosynthetic pigments Electrolyte leakage Photosynthetic efficiency Total glutathione content Leaf disc senescence assay Superoxide dismutase (SOD) activity Glutathione reduction activity Catalase activity For the evaluation of the drought-stress responses by the plants under field, pot, and in vitro conditions, the following analysis should be conducted:In comparison to visual measurement, the reliability and accuracy of stress evaluation have increased with the application of imaging tools and the standardization of visual assessments. However, a large number of wild banana species are still poorly characterized and not evaluated for important new allelic diversity for stress tolerance. In a recent study, dynamic high-throughput phenotyping, modeling, and validation techniques were employed to evaluate genotype-dependent transpiration responses to variable environmental conditions in diverse wild banana genotypes and subspecies containing known ancestors of banana cultivars . The higScientific and information technology advancements are now being used to generate and process large biological data sets derived from omics studies. The various applications of “omics tools,” such as genomics, proteomics, and transcriptomics, have promoted a critical shift in the study of biological sciences. Furthermore, genomics and bioinformatics approaches have greatly enhanced our understanding of the genetic diversity of several banana wild relatives and their links to the desired phenotypic traits. Moreover, to describe the cellular phenotype and establish a connection with the distinct banana genotype, the combination of RNA-Seq, proteomics, and metabolite studies is very helpful .Information produced by the genomics of CWRs facilitates their use to increase the genetic variability of crop plants. The efficient genome sequencing of CWRs and their effective usage in agricultural improvement are made possible by advancements in DNA sequencing technology . With thhttps://banana-genome-hub.southgreen.fr/, accessed on 22 December 2022) provides the modules to facilitate the integration of various systems that include several major banana datasets [https://www.biodiversityinternational.org/banana-genebank/, accessed on 22 December 2022) are helping to explore the banana resources for their genetic variability. The ITC is the world’s largest collection of banana germplasm (1617 accessions of edible and wild species of banana from 38 countries) and was established with the support of the FAO to secure the long-term conservation of the entire banana gene pool for the benefit of future generations. It also ensures that germplasm is free from pests and diseases and available under the International Treaty on Plant Genetic Resources for Food and Agriculture. It also acts as a crucial safety backup and transit center for national banana genebanks. The ITC has provided more than 18,000 accession samples of bananas during its more than 35-year existence to researchers and farmers in 113 countries [M. acuminata was compared with the NAC genes of Arabidopsis thaliana, Vitis vinifera, and Oryza sativa [The Banana Genome Hub , and several dehydrogenases are involved in NAD/NADH homeostasis and respiration [Musa accession of northeast India to understand genotype-specific stress tolerance. Two-dimensional gel electrophoresis has an important application in studying the differential expression of stress-responsive proteins [Using an in vitro growth model, the screening of drought-tolerant varieties and their mechanisms in different piration . Thus, tproteins ,133. Furproteins . Using hproteins .The identification and characterization of drought tolerance genes provide a huge opportunity for successful banana genetic improvement through the intervention of biotechnological advancements. To create designer crops with desired features, biotechnological techniques, such as gene editing or genome editing, are extensively used ,135,136.Streptococcus pyogenes adaptive immune system [Among such editing technologies, CRISPR/Cas-mediated tools have emerged as the most potent and precise methods for crop genome editing, originally derived from the e system . The CRIe system . Additioe system , and base system ,142. Thee system . The roue system ,144,145,e system ,146, ande system . In the e system ,150,151.e system ,53. Prote system ,153.lycopene epsilon-cyclase to develop the β-carotene-enriched Grand Naine (AAA) banana [CCD4 gene in β-carotene accumulation in Rasthali fruit pulp, demonstrating the transgene-free approach to banana genetic improvement [Despite all the difficulties associated with genetic transformation and regeneration in bananas, several research groups have successfully optimized and executed research studies by targeting some marker genes, such as phytoene desaturase (PDS), to demonstrate the proof of concept of genome editing ,154. The) banana . Similar) banana . Further) banana ,156,157.) banana  demonstrrovement . As a rerovement ,160,161.rovement ,163. In CWRs are closely related species of domestic genotypes with a high tolerance towards different stress conditions and, thus, need to be thoroughly explored for different traits ,165. Ban


Sanguisorba officinalis  exerts significant anti-proliferative activities against colorectal cancer (CRC) cells. Since most of CRC result from the aberrant activation of the Wnt/β-catenin signaling pathway, inhibitors of the Wnt pathway are considered as promising anti-CRC agents. This study aimed to investigate whether DY could be a potential herbal Wnt inhibitor, and the bioactive constituents and underlying molecular mechanisms for DY's inhibiting activities would be studied as well. Accordingly, the inhibitory activities of DY and its main components against the Wnt pathway were assessed using the single-luciferase reporter assay based on HEK293 cells. Additionally, the levels of key Wnt-related genes or proteins were measured to verify the inhibitory effects on the Wnt pathway of CRC cells. Finally, the underlying mechanisms accounting for the efficacy of candidate drugs were explored by the transcriptomic study. Results show that DY and its tannins (RZ), and saponins (ZG) significantly inhibited the Wnt pathway of HEK293 cells activated by wnt3a. However, their respective constituents were not effective as expected. Additionally, DY and RZ prominently down-regulated the levels of β-catenin and Wnt-targeted genes including Axin2, c-Myc or CyclinD1 of three CRC cells. Transcriptomic profiling study suggests that the down-regulation of the mRNA levels of Wnt-related genes such as LPAR6 may be associated with the inhibitory effects of DY and RZ on the Wnt pathway of HT29 cells. Therefore, our studies first uncovered the blocking activity of DY on the Wnt pathway, providing evidence for the rationale of developing Wnt inhibitors from DY as anti-CRC agents. Sanguisorba officinalis  exerts significant anti-proliferative activities against colorectal cancer (CRC) cells. Proteins, namely APC, Axin, CK1 and GSK3β, are the main components of this complex, which cooperate with each other to avoid activating the Wnt pathway by degrading the cytoplasmic β-catenin.3 Mutation of the tumour-suppressor genes or oncogenes such as APC, Axin2, SMAD4 or KRAS would cause degradation of the complex and allows β-catenin accumulated in the cytoplasm to transfer into the cell nucleus. Then Wnt/β-catenin signaling is prone to be activated when nucleus β-catenin integrating with TCF/LEF transcription factors.4,5 Subsequently, through activation of the TCF encoding genes, Wnt pathway activators would result in the progression and metastasis of CRC.6 Therefore agents exerting inhibitory activities towards Wnt/β-catenin pathways are potentially anti-CRC medicines. Up to now, a great number of Wnt signaling inhibitors have been discovered, most of which are small molecule compounds or antibodies. Although some of them exhibit significant inhibitory effects (IC50 LGK947 = 0.4 nM),7,8 their cytotoxicity remains a critical issue to be concerned.9The pathogenesis of CRC is complex and is still not clearly understood. However, based on current reports, the aberrant activation of the Wnt/β-catenin pathway is believed to be a major factor contributing to the progress of CRC.10 white hellebore,11Tripterygium wilfordii12 and Coptis chinensis13 were found to exert potential anti-CRC activities. However, as the inhibitory effects of these herbs against the Wnt pathway were not strong enough,14 their anti-cancer effects were unsatisfactory, indicating that it is important to screen and discover new herbal Wnt inhibitors with higher inhibitory activities for clinical use.In addition to western medicines, Chinese medicines have also achieved satisfactory efficiencies in treating human cancers. Compared to the western medicine, Chinese medicines have lower cytotoxicity and much more therapeutic targets, enabling them to be a better source of anti-CRC agents. Therefore, herbal Wnt inhibitors are also worthy being developed as potential anti-CRC therapeutics. Recent studies reported that some traditional Chinese medicines such as ginseng,Sanguisorba officinalis is a traditional Chinese medicine that has been widely used in the folk for treating gastrointestinal diseases. Our previous study found that a water extract of DY exhibited strong antiproliferative activities against CRC cells. Based on the close association of CRC with the Wnt/β-catenin pathway, we hypothesized that DY may exhibit an inhibitory effect towards Wnt signaling. Therefore, the aims of this study are to verify the inhibitory effect of DY against the Wnt pathway and to investigate the underlying mechanism. In addition, the potential active component of DY that's responsible for the inhibition will be studied as well.2.2.1Sanguisorba officinalis L. radix  are listed in Table s1.15DY is widely planted around China. We only selected its genuine medicinal materials for our study. The origins and voucher numbers of the 7 batches of 2.2Gallic acid , ellagic acid  and catechinic acid  were from Chengdu PUSH Bio-technology Co., Ltd. . Ziyuglycoside I (98%) and ziyuglycoside II (98%) were purchased from Chengdu PureChem-Standard Co., Ltd . DEPC solution obtained from Sangon Biotech . Wnt3a protein was purchased from EMPOWERING STEM CELL R&D . The CCK-8 kit was obtained from Dojindo  and Steady-Glo® Luciferase Assay System was from Promega Corporation . PrimeSCript™ RT reagent Kit with gDNA Eraser and SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) were purchased from Takara Bio Inc . RIPA reagent, Anti-β-catenin (#19807) and anti-β-actin (#4970) were from Cell Signaling Technology, Inc. . Alexa Fluor 594 AFFINIpure Goat Anti-Rabbit IgG (H + L) and DAPI Fluoromount-Gtm were obtained from YEASEN Co., Ltd .2.32.To first evaluate the activity of DY on the Wnt/β-catenin pathway, we used a stable TCF reporter cell line based on HEK293 cells obtained from Curegenix Inc. . And HT29 human colorectal cancer cells were kindly gifted by Prof. Qing Li . Both cell lines were cultured in DMEM containing 1% penicillin–streptomycin and 10% fetal bovine serum  in the incubator at 37 °C in 5% CO2.44 cells per well) for 24 h, which were then treated with DMEM medium supplemented with the water solution of DY or its constituents. After 24 h, cells were processed by CCK8 reagents (10 μl) according to the instructions of commercial kit and their viabilities were measured on a microplate reader  at 450 nm.HEK293 and HT29 cells were seeded in 96-well plates (102.54 cells per well for 24 h. Then cells were co-treated with wnt3a (100 ng ml−1) and DMEM medium with DY or its constituents for another 24 h. After that, cells were cultured with Steady-Glo@ assay reagent (30 μl per well) on the orbital shaker plate for 5 min. The mixed solutions were accordingly transferred to the HTS Transwell®-96 Receiver plates , and Monochromators Based Multimode Microplate Reader  was loaded to detect the activities of the Wnt-based luciferase.The HEK293 cells were seeded in the 96-well plates at the density of 2 × 102.65 cells per well) were respectively treated with wnt3a (control group), DY + wnt3a, RZ + wnt3a and ZG + wnt3a for 24 h, which were then digested by trypsin for 10 seconds and washed twice with PBS solution. By centrifuging 3 min at 3000 rpm, the cell pellets were incubated with 100 μl RIPA reagent on ice for 30 min to obtain the cellular lysates. The lysates were centrifuged at 10 000g for 20 min at 4 °C. The supernatant was collected as the total protein extract, the concentration of which was analyzed using the bicinchoninic acid (BCA) method. 30 μg of protein sample was loaded on 8% gel to perform the sodium dodecyl sulphate-polyacrylamide gel electrophoresis, which was then transferred to PVDF membranes . After blocking with 5% skim milk powder solution (2 h), the transfected films were blotted with the anti-β-catenin and anti-β-actin for 2 h, following by 1 h of HRP-conjugated anti-rabbit secondary antibody incubation. Finally, the targeted protein bands were visualized and imaged by the ChemiDoc XRS+ system .Cells  at 260/280 nm. Subsequently, 10 μg total RNA was used to synthesize the complementary DNA (cDNA) based on the manufacturer's instructions of PrimeSCript™ RT reagent Kit. In brief, the gDNA of the RNA samples was first eliminated using 2 μl gDNA eraser buffer and 11 μl gDNA eraser. 10 μl of the gDNA-free RNA was then added into the reverse-transcription system  to produce the cDNA . With 2 μl cDNA (<100 ng), other reaction reagents in the SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) Kit including 12.5 μl SYBR Premix Ex Tap II, 1 μl PCR Forward/Reverse Primer and 8.5 μl dH2O were mixed according to the kit's instructions. The primers sequences used were: Axin2, forward, 5′-AGGCTAGCTGAGGTGT-3′, and reverse, 5′-AGGCTTGGATTGGAGAA-3′; DKK-1, forward, 5′-CTGCAAAAATGGAATATGTGT-3′, and reverse, 5′-CTTCTTGTCCTTTGGTGTGA-3′; c-Myc, forward, 5′-CCACACATCAGCACAACTACG-3′, and reverser, 5′-CCGCAACAAGTCCTCTTCAG-3′; CyclinD1, forward, 5′-TCGGGAGAGGATTAGGTTCC-3′, and reverse, 5′-GTCACTGGATGGTTTGTTGG-3′; FGF20, forward, 5′-ATTCATCAGTGTGGCAGTGG-3′, and reverse, 5′-GCTCCCTAAAGATGCATTCG-3′; GAPDH, forward, 5′-AGGTCGGAGTCAACGGATTTG-3′, and reverse, 5′-TGTAAACCATGTAGTTGAGGTCA-3′.Cells were treated in the same way as described in western blotting assay. After drug treatment, total RNA from cell samples were extracted. In brief, each sample was first lysed by 0.5 ml Trizol reagent , and then 100 μl chloroform was added followed by one-minute vortex. Collected the supernatants after 10 minutes centrifugation at 12 000et al.16 Briefly, the amplification conditions were 30 s at 95 °C, 40 cycles of 5 s at 95 °C and 30 s at 60 °C, which was performed on Bio-Rad PCR system . The obtained data were analyzed by the ΔΔCt approach.For the procedures of qPCR please refer to the methods described in Bae's, 2.84 cells per well) were treated with DY, RZ or ZG for 24 h, which were then washed once with PBS solution. The cells were fixed with 4% paraformaldehyde for 30 minutes. Then the cells were washed in PBS solution and permeabilized with 0.1% Triton X-100. Blocked in 5% skim milk solution (30 min), the cells were blotted with anti-β-catenin and anti-β-actin for 2 h, after cells incubating with Alexa Fluor 594 AffiniPure Goat Anti-Rabbit IgG (H + L) for 1 h, they were washed with PBS and then mounted with DAPI Fluoromount-G™. Finally, the cells were observed and imaged by the confocal microscopy .Cells  were treated DY, RZ and ZG solutions for 24 h. The samples were then washed and lysed by 1 ml TRizol reagent. The transcriptomics analysis was accomplished by our collaborator, Biomarker Technologies . The analytic steps were as follows: at first, 1% agarose gels were used to monitor RNA degradation and contamination. RNA purity was assessed on NanoPhotometer spectrophotometer . And the concentration and integrity of RNA were respectively measured by Qubit RNA Assay Kit in Qubit 2.0 Fluorometer  and RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system . Then, RNA sequencing samples were prepared based on 1 μg RNA per sample. NEBNext UltraTM RNA Library Prep Kit for Illumina  was used to establish sequencing libraries. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads, which was then fragmented using divalent cations. First strand cDNA was synthesized by using random hexamers primer and M-MuLV Reverse Transcriptase (RNase H-), and the second one was then synthesized by DNA polymerase I and RNase H. After a further modification of the synthesized cDNA, PCR was performed and their products were purified and assessed on the Agilent Bio-analyzer 2000 system. Afterwards, based on the established library, an Illumina Hiseq 2500 platform was applied to conduct the sequencing, and paired-end reads were generated. The depth of sequencing coverage was 10× and the length of sequence reads was from 200–250. In this study, at least 6 Gb clean data were obtained for each sample. Before data analysis, raw data were processed by removing reads containing adapter, ploy-N and low quality. Only the clean data with a high match  or only one mismatch was further analyzed and annotated based on the reference genome (GRCH38) were subjected to further analysis by Tophat2 tools software. Differential expression analysis  of two samples was conducted on the DEGseq (2010) R package. Q value < 0.005 and |log 2 (fold change)| ≥ 1 was set as the threshold for significantly different expression. GO enrichment analysis of DEGs was performed on GOseq R packages (http://www.geneontology.org/) and KEGG pathway analysis of DEGs was implemented by the KOBAS 3.0 software.Prior to the sequencing analysis, HT29 cells . P < 0.05 demonstrated a statistically significant difference.In addition to the transcriptomics study, all experiments have repeated thrice and their results were expressed as mean ± SD. For the statistical analysis, one-way ANOVA approach based on Kruskal–Wallis test was performed, which was followed by 3.3.115 were listed in Fig. s1B50 value at 2.20 ± 0.74 μg ml−1  were studied to confirm the Wnt-inhibiting effects of the indicated medications on CRC cells. Based on Axin2 levels in HCT116 cells and showed a tendency of down-regulating Axin2 and c-Myvc in RKO cells, which further verified its inhibitory effects on Wnt signaling pathway of CRC cells. Other genes were not significantly influenced. Western blot results are not available here, because no blots of β-catenin were observed in the two CRC cell lines.The activation of Wnt/β-catenin pathway is closely related to the incidence of colorectal cancer.29 cells  and s2B†ch other . In termCyclinD1 . RZ mereCyclinD1  are over3.3https://figshare.com/s/c20cae72a30e6551c962, including function annotation, GO analysis, GO tree analysis, KEGG pathway analysis and so on. Table s3 and Fig. s4Transcriptomic profiling study is a preferred strategy to find out the molecular mechanisms of certain biological events based on a holistic view. We took use of this high-throughput technology to systematically explore the underlying mechanisms accounting for the inhibitory effects of DY and its main components on the Wnt pathway of CRC cells-HT29 cell. The RNA-seq data can be referred to the link Vibrio cholerae infection, toxoplasmosis, HTLV-I infection and hepatitis C were obviously regulated by RZ as well. As reported, tannins exerted anti-proliferative activities to many cancer cell lines such as MCF-7, Caco-2 and DU145,30 and it could effectively treat kinds of human diseases including inflammation, Type II diabetes mellitus and leukemia.31–33 Hence, the KEGG results basically conform to the reported facts. In the ZG group, all the DEGs were enriched in metabolic pathways, namely, ABC transporters, purine metabolism and drug metabolism-other enzymes. Furthermore, the above findings were confirmed using statistical analysis of pathway enrichment  analysis was further carried out to explore the potential functions of the DEGs. Fig. s7t Fig. s8.3.434 In terms of Wnt signaling pathway, DY and RZ only significantly regulated a few of genes, while no DEGs were observed in the ZG group , FGF19 & DUSP10 (MAPK), STAT1 & IRF9 (Jak-STAT) and RASSFS (Ras) were down-regulated, while LAMC2 (PI3K-Akt), DUSP1 & DUSP5 (MAPK), PPARD (PPAR) and PTCH2 (Hedgehog) were up-regulated. As reported, the above genes played important roles in the hyperactivation of Wnt pathway and they were also found generally over-expressed in the Wnt-activated CRC.35–41 Therefore, current findings suggest that the inhibitory activities of DY and RZ towards the Wnt/β-catenin signaling pathway might be relevant to their down-regulation of the indicated genes, which should be further confirmed. Taken together, DY and RZ can effectively down-regulate the key protein and genes levels in the Wnt signaling pathway. As colon cancer is highly associated with the abnormal activation of Wnt pathway and DY and RZ were able to significantly inhibit Wnt pathway, we hypothesize that they exhibit anti-CRC potentials, which would be further verified in our future work.Based on the findings that DY and RZ significantly regulated genes involved in cancers, we analyzed the DEGs on the cancerous pathways, especially the Wnt/β-catenin signaling pathway and its cross-talking pathways including Notch, PI3K-Akt, MAPK, Jak-STAT, PPAR, Ras and Hedgehog.ZG group . For theand ASPM . Moreove4.LPAR6, PRICKLE4, ASPM, MYB, FGF19 and STAT1. All in all, our findings suggest that DY and might be a good resource of Wnt inhibitors. As CRC is highly associated with the aberrant activation of Wnt pathway, DY is also possible to be developed as anti-CRC agents when supported by more pre-clinical and clinical evidence, which will be studied in the near future.In conclusion, the inhibitory effects of an aqueous extract of DY on the Wnt/β-catenin signaling pathway of HEK293 is first reported. The study of bioactive constituents demonstrates that RZ and ZG of DY played important roles in inhibiting Wnt pathway, while their representative constituents were not the active as expected. The active compounds in DY are still under investigation in our lab. DY and RZ also could significantly inhibit the Wnt pathway in CRC cell line, HT29, which might be resulted from their down-regulation of Wnt-related genes such as None declared.RA-008-C8RA00438B-s001


Motor neuron (MN) diseases, including amyotrophic lateral sclerosis, progressive bulbar palsy, primary lateral sclerosis and spinal muscular atrophy, cause progressive paralysis and, in many cases, death. A better understanding of the molecular mechanisms of pathogenesis is urgently needed to identify more effective therapies. However, studying MNs has been extremely difficult because they are inaccessible in the spinal cord. Induced pluripotent stem cells (iPSCs) can generate a theoretically limitless number of MNs from a specific patient, making them powerful tools for studying MN diseases. However, to reach their potential, iPSCs need to be directed to efficiently differentiate into functional MNs. Here, we review the reported differentiation protocols for spinal MNs, including induction with small molecules, expression of lineage-specific transcription factors, 2-dimensional and 3-dimensional cultures, as well as the implementation of microfluidics devices and co-cultures with other cell types, including skeletal muscle. We will summarize the advantages and disadvantages of each strategy. In addition, we will provide insights into how to address some of the remaining challenges, including reproducibly obtaining mature and aged MNs. Amyotrophic lateral sclerosis, progressive bulbar palsy, primary lateral sclerosis, and spinal muscular atrophy are motor neuron (MN) diseases characterized by progressive degeneration of MNs, resulting in the loss of voluntary and involuntary movement . Since tMNs control movement by integrating signals from the brain and sensory systems . Upper MOne of the most significant challenges in studying these diseases is that patient MNs are located within the brain (upper MNs) and spinal cord (lower MNs), making them only accessible in post-mortem tissue. However, the MNs of most interest for protecting are not available for analysis in post-mortem tissue because they degenerate during the disease course. Thus, the earliest stages of pathogenesis, which might be amenable to a therapeutic intervention, occur while these MNs are hidden from view. Therefore, another model system is needed to recapitulate early MN pathogenesis using living human MNs.Induced pluripotent stem cells (iPSCs) are a powerful tool for understanding human MN biology and disease pathogenesis, particularly in their earliest stages. Takahashi and Yamanaka demonstrated that the expression of four transcription factors, OCT4, SOX2, KLF4, and C-MYC, reprograms somatic cells into iPSCs  that havReaching their transformative potential requires efficient and consistent differentiation of iPSCs into functional MNs. Many labs have contributed to the development of increasingly sophisticated differentiation protocols to obtain iPSC-derived MNs, including the use of specific small molecules to recapitulate developmental morphogen signals , expressIt is important to match the research question with the appropriate model. Many protocols, particularly in 2-dimensional cultures, focus on obtaining homogeneous populations of MNs, and these cultures are particularly useful for studying the development of MNs as well as cell-autonomous disease pathology. However, a different strategy would be needed to understand more complex systems such as motor circuits and non-cell autonomous disease pathogenesis, including prion-like spreading, which would require reproducible heterogeneous cultures. Here, we review reported differentiation protocols for obtaining spinal MNs, summarize the advantages and disadvantages of each strategy, and discuss possible solutions for the remaining challenges.in vitro , in combination with RA  and are relatively expensive. Moreover, PDMS, which is commonly used to fabricate microfluidics chips can absorb molecules such as fluorescent dyes, making imaging challenging. The combination of PDMS with polytetrafluoroethylene has been proposed to reduce this problem . Materia3D cultures are heterogeneous and, unlike 2D cultures, cannot be easily integrated into existing automation systems and massively parallel screening campaigns. Compared with animal models, organoids and organ-on-a-chip could enable the study of human diseases at a lower cost with fewer ethical concerns assuming that issues of scalability and reproducibility are addressed successfully. One example are the neuromuscular organoids generated by Faustino and colleagues, which would be an ideal platform for studying NMJ diseases and identifying novel drug candidates .However, there are multiple challenges when using organ-on-a-chip and organoids, including difficulties in reproducibility, resulting in considerable variability in their development, cellular composition and, in the case of organoids, organization between batches and, sometimes, organization between organoids within one experiment . This heMicropatterned arrays can be used as templates for generating organoids. For example, creating an artificial organizing center by embedding a mass of cells expressing a certain morphogen at one pole of the organoid  or usingTo reduce variability, 3D cultures need to overcome the complication of ensuring that the factors added to the culture medium are administered equally to all cells within the 3D structure. Cells within 2D cultures are widely accessible to the culture medium, enabling uniform exposure. The approach suggested by Another approach is using gelatin-based 3-D bioprinted bioink scaffolds. in vitro screening platform for brain-penetrating agents . Fusion organoids cultured for 40 days contained functional blood–brain barrier-like structures as well as microglial cells. Thus, in addition to potentially enabling the cultivation of larger organoids, this model also allows the modeling of interactions between neurons and non-neuronal cells, particularly the vasculature and microglia niche . iPSC-derived neurons did not retain aging-associated gene signatures. In contrast, iNs displayed age-specific transcriptional profiles and revealed age-associated decreases in the nuclear transport receptor RANBP17. Using RNA sequencing, they showed that RANBP17 levels decreased with age . This coInterestingly, it is possible that disease might be associated with aberrant expression of proteins required for healthy aging. For example, NRSF/REST is a feature of normal aging in human cortical and hippocampal neurons; its expression is low in “young” neurons and high in “aged” neurons . Howeverin vivo. As summarized in in vitro.iPSCs are powerful tools for modeling MN diseases, but doing so requires large quantities of functional MNs having the same characteristics, age, and functional connections as those found in patients. There has been considerable success in developing protocols to direct the differentiation of iPSCs into MNs, which can now be obtained near homogeneity within 1–2 weeks. Recent years, have seen a considerable increase in the diversity and sophistication of MN cultures, each marking an improvement in replicating human MNs and physiological function


The canonical Wnt/β-catenin signaling pathway plays crucial roles in many biological processes including neurodevelopment, cell survival, and cell cycle regulation, as well as microglial activation, thereby potentially presenting one of the key targets of Cd2+ neurotoxicity. Therefore, in this follow-up study, we investigated the implication of the Wnt/β-catenin signaling pathway in Cd2+-induced developmental disorders and neuroinflammation and revealed that environmental Cd2+ exposure significantly affected the expression of key factors in the zebrafish Wnt/β-catenin signaling pathway. In addition, pharmacological intervention of this pathway via TWS119, which can increase the protein level of β-catenin and act as a classical activator of the Wnt signaling pathway, could significantly repress the Cd2+-induced cell cycle arrest and apoptosis, thereby attenuating the inhibitory effects of Cd2+ on the early development, behavior, and activity, as well as neurodevelopment of zebrafish larvae to a certain degree. Furthermore, activation and proliferation of microglia, as well as the altered expression profiles of genes associated with neuroimmune homeostasis triggered by Cd2+ exposure could also be significantly alleviated by the activation of the Wnt/β-catenin signaling pathway. Thus, this study provided novel insights into the cellular and molecular mechanisms of Cd2+ toxicity on the vertebrate central nervous system (CNS), which might be helpful in developing pharmacotherapies to mitigate the neurological disorders resulting from exposure to Cd2+ and many other environmental heavy metals.Cadmium (Cd) is a toxic heavy metal and worldwide environmental pollutant which seriously threatens human health and ecosystems. It is easy to be adsorbed and deposited in organisms, exerting adverse effects on various organs including the brain. In a very recent study, making full use of a zebrafish model in both high-throughput behavioral tracking and live neuroimaging, we explored the potential developmental neurotoxicity of Cd Many cod rivers ,8,9,10. d rivers ,12. Thisd rivers  has beend rivers ,15. Due al cells ,17,18, tal cells ,19,20. Iells [2+ ,22,23,24 signals . The Cd2diseases ,27,28. I2+ exposure could be closely related to the Wnt/β-catenin signaling pathway. However, knowledge of this crucial pathway and its role in the Cd2+-caused neurotoxicity is still very limited.Among the various molecular components and pathways involved in neurodevelopment, the Wnt/β-catenin signaling pathway is essential and has been extensively investigated ,30. Brie2+ exposure. Importantly, we revealed associations between Cd toxicity and neurodevelopmental disorders as well as microglia-mediated neuroinflammation [2+-induced neurodevelopmental and neurological disorders. The results of this study may not only provide novel insights into the cellular/molecular mechanisms of Cd2+ toxicity on vertebrate CNS, but also be very helpful to exploiting pharmacotherapies to mitigate the neurological disorders resulting from exposure to environmental Cd, as well as many other environmental heavy metals.Zebrafish, as a classical vertebrate model system with a well-established genetic and developmental background, possesses significant advantages including strong fertility, transparent embryos, small size, rapid development, convenient drug delivery, and importantly, sufficient homology with humans at the genetic level. Furthermore, various well-constructed transgenic lines expressing fluorescent proteins offer powerful tools for behavioral tracking of diverse cell types ,44. Therammation . However2+ exposure on the Wnt/-catenin signaling pathway, we examined the expression levels of Wnt-related proteins and genes in 6 dpf zebrafish larvae exposed to different levels of Cd2+. Six days after Cd2+ exposure, the content of β-catenin significantly decreased with the increase in Cd2+ levels , endogenous inhibitor ddk1, and target genes  were determined by qRT-PCR analyses. The results showed that Cd2+ exposure significantly downregulated the expression of wnt4a, wnt10b, β-catenin, lef1, axin2, myca, ccnd, and sp52, while it upregulated the expression of ddk1 and gsk3β  and an inhibitor (XAV939) of the Wnt signaling pathway to the culture medium of zebrafish larvae coexposed to 200 μg/L of Cd2+ and continuously analyzed their early developmental indicators at different stages up to 7 dpf  zebrafish line was applied and imaging was performed initially under the standard fluorescence microscope. The results showed that the fluorescence intensity of the brain and spine region in the 6 dpf larvae decayed in the Cd2+-exposed individuals, whereas this effect was significantly attenuated in the Cd2+ and TWS119 coexposed larvae , nestin, α1-tubulin, syn2a, and gap43, which are closely involved in neuron development and differentiation, were detected by qRT-PCR analysis. As expected, the expression of these genes was significantly downregulated in Cd2+-exposed 6 dpf larvae, while this trend was remarkably reversed upon activation of the Wnt signaling pathway by TWS119  zebrafish larvae were selected, and their cell cycle distributions were determined by using flow cytometry. Our results showed that Cd2+ exposure significantly reduced the number of cells in the G1/G0 phase, increased the proportion of cells in the G2/M phase, and remarkably blocked cells in the S phase, which was highly consistent with our previous results, indicating that Cd2+ exposure leads to neuron-related cell cycle arrest. However, upon activation of the Wnt signaling pathway via TWS119 coexposure, the proportion of cells in the G1/G0 phase increased to a similar level as the normal control group, while the blockage of cells in the G2/M phase and the S phase was to some extent released  transgenic line, we observed and verified that upon Cd2+ exposure, the microglia in the brain of zebrafish larvae showed distinct branch retraction, changing their morphology from ramified to amoeboid, which is a typical morphological change of microglia activation and neuroimmune response. Compared with the Cd2+ exposed larvae, the microglia in the Cd + TWS119 group tended to possess longer branches and were more like those in the normal control group, while the microglia in the Cd + XAV939 group remained amoeboid , as a toxic and ubiquitous heavy metal pollutant, is easily accumulated in organisms, and brings great risks to human health and ecosystems. In recent decades, Cd has been widely recognized as one of the neurotoxic agents associated with many neurological diseases. It may alter the release of neurotransmitters and lead to changes in the blood–brain barrier (BBB) by inducing oxidative stress . It may 2+ on the early development, structure, and function of the CNS [2+-induced neurotoxicity. Briefly, in this study, we focused on the implications of the Wnt/β-catenin signaling pathway, and revealed that (1) Cd2+ exposure significantly affected the expression of key factors in the zebrafish Wnt/β-catenin signaling pathway; (2) pharmacological activation of the Wnt/β-catenin signaling pathway significantly repressed the Cd2+-induced cell cycle arrest and apoptosis, thereby attenuating the inhibitory effects of Cd2+ on early development, behavior, and activity, as well as the neurodevelopment of zebrafish larvae; (3) activation and proliferation of microglia, as well as the altered expression profiles of genes associated with neuroimmune homeostasis triggered by Cd2+ exposure, could also be significantly alleviated by the activation of the Wnt/β-catenin signaling pathway.Zebrafish is a famous model system in the study of genetics and developmental biology ,55. Beca the CNS , and on 2+ impairs the early neurodevelopment of zebrafish by inducing cell cycle arrest and apoptosis [2+ to the CNS development of zebrafish might be closely related to the Wnt/β-catenin signaling pathway.Numerous articles reported that the Wnt/β-catenin signaling pathway plays crucial roles in many biological processes including neurodevelopment, cell survival, and cell cycle regulation, as well as microglial activation ,58,59. Fpoptosis ; therefo2+ inhibited the Wnt/β-catenin signaling pathway, which is contrary to the traditional view that Cd activates the Wnt/β-catenin signaling pathway and induces cancer [2+-exposure-induced renal fibrosis and/or cancer might be mediated by the activation of the Wnt signaling pathway [2+-induced neurotoxicity by adding an activator (TWS119) and an inhibitor (XAV939). Our results showed that the early developmental impairments induced by Cd2+ exposure in zebrafish embryos/larvae were significantly reduced when Wnt activators were added. Neuroimaging of the transgenic zebrafish Tg(elavl3: EGFP) line showed that activation of the Wnt/β-catenin signaling pathway could also reverse the nerve damage caused by Cd2+ exposure. Consistently, Miryam A. Fragoso et al. reported that the Wnt signaling pathway protects retinal ganglion cells from damage caused by high pressure, oxidative stress, and hypoxia [2+- induced microglia activation by using the Tg(ApoE:EGFP) line. The results showed that activation of Wnt signaling pathways could indeed significantly reduce the degree of microglia activation and neuroinflammation in the brain. Thus, our results indicated that the Wnt/β-catenin signaling pathway is strongly implicated in the developmental neurotoxicity and neuroinflammation induced by Cd exposure, but whether the Wnt signaling pathway is a direct target of Cd still needs further investigations.Interestingly, in our study, the relatively high concentrations of Cds cancer ,66,67. W pathway ,69. Yifa pathway . On the  pathway . Therefo hypoxia . The rol hypoxia ,74,75. R hypoxia . Therefo2) was purchased from Sigma-Aldrich. The 1 mM stock solution was prepared in deionized water and stored at −20 °C. The working solution was obtained by diluting the stock solution in E3 embryo medium . To modulate Wnt signaling activities, zebrafish embryos/larvae were treated with the inhibitor XAV939  and activator TWS119 . Tricaine  and 1-pheny1–2-thio-urea  were purchased from Sigma-Aldrich .Cadmium chloride (CdClTg(elavl3:EGFP) line [Tg(ApoE:EGFP) line [Zebrafish husbandry and experimental procedures were reviewed and approved by the Ethics Committee of the School of Life Sciences, Lanzhou University . A great deal of effort was made to minimize animal suffering. The AB (wild-type) line and transgenic FP) line  were obtFP) line  was giftFP) line ,79.2, or in combination with Wnt agonist (3 μM TWS119)/inhibitor (3 μM XAV939) suspended in E3 medium, which was refreshed daily. The morphological characteristics of zebrafish embryos/larvae were recorded under the stereomicroscope at different developmental stages ranging from 2 hpf to 144 hpf. Briefly, mortality as well as other parameters including hatchability, body length, cardiac rate, and malformation of embryos/larvae were recorded as previously reported [Newly-hatched zebrafish embryos were collected and washed with standard E3 embryo medium within one-hour postfertilization, and then were examined under the stereomicroscope . Normally developing embryos were exposed to CdClreported . In addireported .To start, 6 dpf zebrafish larvae without any observable morphological abnormalities were randomly selected from each group for neurobehavioral tests, as previously described ,81. Brieg, 4 °C for 10 min, and stored at −80 °C. An equal amount of protein (25 μg) determined via BCA assay  was diluted with Laemmli buffer (Bio-Rad) containing 2-mercaptoethanol (Sigma-Aldrich). The Western blotting was performed by using a standard protocol [The 6 hpf zebrafish larvae (×100 per sample) were collected and homogenized in RIPA Buffer  containing protease inhibitors . Samples were centrifuged at 10,000× Tg(elavl3:EGFP) and Tg(ApoE:EGFP) zebrafish lines were used for in vivo imaging of neurons and microglia, respectively, located in the brain region of zebrafish larvae. Briefly, larvae without any morphological abnormalities were washed with E3 buffer, anesthetized by using 0.01% MS-222 (Sigma-Aldrich), and then were immobilized in 1% low melting-point agarose, with their head region exposed. Imaging of neurons in the Tg(elavl3:EGFP) larvae were initially conducted by using a standard epifluorescence microscope (Olympus BX51). Furthermore, the two-photon confocal laser scanning microscope (Olympus FV1000) was used to image the fine structure and morphology of neurons and microglia in the telencephalon and optic tectum region. All the imaging data were processed by using the ImageJ software  and were presented in single stack/z-stacks. Three-dimensional reconstruction and morphological analyses of neurons and microglia were performed by using the IMARIS software  based on the user guide.The Cell apoptosis in the brain of zebrafish larvae was detected by staining of acridine orange (AO) . BrieflyTg(elavl3:EGFP) zebrafish larvae  which had been digested and homogenized, were washed twice with ice-cold PBS, and then were fixed in 70% ethanol at 4 °C for overnight. Next, fixed cells were washed with PBS at 4 °C for two times, resuspended in RNase A solution, and then were stained with DAPI . Subsequently, EGFP specifically labeled neurons and neuronal precursors were sorted, and the distribution of cell cycles were determined via the FITC channel by using the FACSCalibur flow cytometer . Finally, the data were processed and analyzed by using the FlowJo Version 7.6.1 software in accordance with the manufacturer’s instructions.Analyses of the cell cycle distribution of neurons and neuronal precursor cells were performed as previously reported ,84. Brie−ΔΔCT method and normalized them according to the relative expression of β-actin.Total RNA was isolated from zebrafish larvae (50 larvae per sample) with the TRIzol reagent  following the manufacturer’s instructions, and cDNA synthesis was performed by using the reverse transcriptional kit . Quantitative real-time PCR (qRT-PCR) was performed with SYBRGreen master mix kit , using the StepOnePlus™  Real-Time PCR system in the Experiment Center of the School of Life Sciences according to the manufacturers’ recommendations. The primer sequences of tested genes are listed in GraphPad Prism 8.0 Software was used to perform statistical analyses in this study. Numerical results are presented in histograms as mean plus standard errors and in boxplots as median and mean, with whiskers showing 90% confidence levels. All data were initially checked for homoscedasticity and normality. In order to determine significant differences, the nonparametric test or analysis of variance (ANOVA) followed by multiple comparison tests were conducted. All the detailed statistical information has been listed in


Ribosomal protein S6 kinase 1 (S6K1), a key downstream effector of the mammalian target of rapamycin (mTOR), regulates diverse functions, such as cell proliferation, cell growth, and protein synthesis. Because S6K1 was previously known to be localized in the cytoplasm, its function has been mainly studied in the cytoplasm. However, the nuclear localization and function of S6K1 have recently been elucidated and other nuclear functions are expected to exist but remain elusive. Here, we show a novel nuclear role of S6K1 in regulating the expression of the Wnt target genes. Upon activation of the Wnt signaling, S6K1 translocated from the cytosol into the nucleus and subsequently bound to β-catenin and the cofactors of the Wnt/β-catenin transcriptional complex, leading to the upregulation of the Wnt target genes. The depletion or repression of S6K1 downregulated the Wnt target gene expression by inhibiting the formation of the Wnt/β-catenin transcriptional complex. The S6K1-depleted colon cancer cell lines showed lower transcription levels of the Wnt/β-catenin target genes and a decrease in the cell proliferation and invasion compared to the control cell lines. Taken together, these results indicate that nuclear S6K1 positively regulates the expression of the Wnt target genes by inducing the reciprocal interaction of the subunits of the transcriptional complex. The Wnt signal transduction pathway is evolutionarily highly conserved in animal species, from Drosophila to Homo sapiens. Wnt signaling plays a central role in various cellular processes, such as cell proliferation, survival, polarity, migration, and differentiation . The abeThe mammalian target of rapamycin (mTOR) is a key regulator of various intracellular and extracellular signaling processes ,11,12. THere, we report a novel role for nuclear S6K1 as a regulator of the Wnt/β-catenin signaling pathway. We found that activation of Wnt signaling leads to S6K1 phosphorylation at T389 and its subsequent nuclear translocation. Nuclear S6K1 bound to β-catenin and other nuclear Wnt transcription cofactors and regulated the expression of the Wnt target genes by affecting the formation of the Wnt transcriptional complex. Consistent with this finding, both the knockdown and inhibition of S6K1 in colon cancer cell lines downregulated the expression of the Wnt target genes and suppressed the proliferation, migration, and transformation of cancer cells. Collectively, the nuclear Wnt/β-catenin signaling in HEK293T cells and colon cancer cell lines is mediated by the nuclear translocation of S6K1.To confirm the relationship between Wnt and mTOR signaling , we examMany studies have reported a possible association between the mTOR and Wnt signaling pathways . HoweverThe nuclear role of S6K1 in regulating transcription has been reported previously ,19. BecaBecause S6K1 regulates the Wnt/β-catenin-mediated transcription, and Wnt-dependent S6K1 activation enhances its binding to β-catenin, we hypothesized that S6K1 could directly phosphorylate β-catenin. However, the in vitro kinase assay showed that S6K1 does not directly phosphorylate β-catenin, whereas it phosphorylates histone H2B as shown in our previous study. a 18]. F. F18]. FNuclear β-catenin activated by Wnt signaling interacts with multiple binding partners, such as T-cell factor 4 (TCF4), E1A-binding protein p300 (p300), Pygopus family PHD finger 2 (Pygo2), and B-cell CLL/lymphoma 9 protein (BCL9), which comprise the transcriptional complex expressing Wnt target genes. Because the stability and localization of β-catenin did not change and were independent of the S6K1 activation b,c, we wPrevious studies have reported the abnormal activation of Wnt signaling in colon cancer cells ,22. In aIn this study, we found that S6K1 is translocated into the nucleus upon Wnt signaling activation and directly binds to β-catenin. Moreover, S6K1 affects the interaction between β-catenin and TCF4, as well as the binding between other Wnt/β-catenin transcriptional cofactors, such as β-catenin, Pygo2, BCL9, and p300, thereby mediating the regulation of the Wnt/β-catenin target gene expression . AlthougHere, we found that long-term treatment with PF-4708671, the specific inhibitor of S6K1, decreased the protein levels of Pygo2. If S6K1 phosphorylates Pygo2 directly, it can be hypothesized that the knockdown or inhibition of S6K1 leads to the reduced phosphorylation of Pygo2 and induces its degradation through increased ubiquitination. Consequently, this downregulation of Pygo2 can suppress the formation of the transcriptional complex that constitutes Pygo2 as a cofactor, thereby reducing the expression of Wnt target genes. To substantiate this hypothesis, further experiments are required to demonstrate that S6K1 can directly phosphorylate and regulate Pygo2.The Wnt/β-catenin signaling pathway regulates many cellular processes, such as cell proliferation, differentiation, and migration. If signal transduction is dysregulated and homeostasis is not maintained, tumorigenesis can occur. Therefore, the Wnt/β-catenin signaling pathway has been targeted for cancer treatment ,27. The We showed that S6K1 translocates from the cytosol into the nucleus after Wnt signaling activation and directly interacts with β-catenin and several Wnt transcriptional cofactors to transcriptionally regulate the Wnt target genes in a kinase activity-dependent manner. Our results revealed that the deregulation of S6K1, which leads to the overactivation of the Wnt/β-catenin signaling, is closely linked to colorectal cancer cell proliferation and migration. It has been well established that the aberrant expression of Wnt target genes induces cancer progression. Therefore, elucidating the detailed mechanism of S6K1 deregulation might be beneficial in developing S6K1-based therapeutic approaches for colon cancer.Anti-p70 S6K1 , anti-phospho-p70 S6K1 T389 , anti-Flag , anti-c-Myc , anti-β-catenin , anti-acetyl-β-catenin K49 , anti-TCF4 , anti-BCL9 , anti-Pygopus2 , anti-p300 , anti-cyclin D1 , anti-S6 , anti-histone H3 , anti-lamin A/C , anti-α-tubulin , anti-actin , and anti-IgG  were used.The following reagents and plasmids were used: pRK5 Myc-tagged human S6K1 wild-type (WT), constitutively active (CA), and dominant-negative (DN). pcDNA Flag-tagged human β-catenin. pGEX-4T-1-based bacterial-expressed GST-tagged empty vector and human β-catenin. Rapamycin, the mTOR specific inhibitor ; PF-4708671, the S6K1 specific inhibitor ; Wnt-3a, Wnt activator that binds to Frizzled ; lithium chloride (LiCl), the GSK-3 inhibitor that acts as a Wnt activator ; and active p70 S6K1 protein for in vitro kinase assay .2 at 37 °C.HEK293T cells were grown in Dulbecco’s modified Eagle medium . HT29 cells were grown in McCoy’s 5A medium (Welgene). CCD-18Co cells were grown in minimum essential medium . DLD-1 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Welgene). LoVo cells were grown in Ham’s F-12K (Kaighn’s) medium . All cells were grown in media supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin . Cells were maintained in a humidified atmosphere of 5% COCells were treated with rapamycin (25 nM) or PF-4708671 (10 μM) to inhibit mTOR or S6K1 activities, respectively, or the same amount of DMSO as a control. Cells were treated with Wnt-3a (300 ng/mL) or LiCl (25 mM) for the indicated time points.g at 4 °C. The supernatant was then transferred into a new tube. For nuclear fractionation, cells were lysed in harvest buffer (10 mM HEPES (pH 7.9), 50 mM NaCl, 0.5 M sucrose, 0.1 mM EDTA, 0.5% Triton X-100, and freshly added DTT, PMSF, and protease inhibitors), incubated on ice for 5 min, and centrifuged at 120× g for 10 min at 4 °C. The supernatant (cytosolic fraction) was transferred into a new tube. The nuclear pellet was washed twice with 500 μL of buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, and 0.1 mM EGTA) and centrifuged at 120× g for 10 min at 4 °C. The supernatant was discarded, and the pellet (nuclear fraction) was resuspended in PRO-PREP for lysis.For immunoblotting, whole-cell proteins were extracted using PRO-PREP protein extraction solution . The cell lysates were centrifuged for 20 min at 16,000× Equal amount of each protein sample was used. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, they were transferred onto polyvinylidene difluoride membranes  using the wet transfer method. The membranes were incubated with the indicated primary antibodies overnight at 4 °C. Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Merck Millipore) for 1 h at room temperature. The HRP signals were detected using AbSignal .3, and 0.1% Triton X-100 in PBS) at room temperature for 30 min. Primary and secondary antibodies were diluted in PBS-BT at an appropriate ratio and incubated at room temperature for 1 h at each step in a humidified chamber. Staining was performed sequentially from the primary to the secondary. Between each step, samples were washed twice with PBS-BT buffer.Cells were cultured in 12-well plate on gelatin-coated cover glass. At 0, 4, and 8 h after LiCl treatment, samples were fixed with 4% paraformaldehyde for 15 min, and rehydrated in PBS for 10 min. Cell permeabilization and blocking process was performed with PBS-BT buffer . The indicated specific antibodies and the agarose beads for the type of antibodies were added to equal amount of each protein sample and incubated at 4 °C overnight. The beads were then centrifuged briefly and washed three times with IP wash buffer (IP lysis buffer without CHAPSO). Immunoprecipitated proteins were eluted with 2× sample buffer  by boiling at 95 °C for 5 min. The eluted protein samples were analyzed by immunoblotting.Cells were harvested and lysed using PRO-PREP  or IP lysis buffer (40 mM HEPES (pH 7.4), 120 mM NaCl, 1 mM EDTA, 50 mM NaF, 1.5 mM Na® FAST qPCR Master Mix . The relative expression levels of the target genes were normalized to the expression levels of GAPDH and β-actin. The primer pairs used for qRT-PCR are listed in Whole-cell RNA was purified using Easy-Blue reagent  according to the manufacturer’s instructions. For cDNA synthesis, 1 μg of purified RNA and Maxime RT premix (iNtRON Biotechnology) were used. qRT-PCR was performed on the CFX96 Real-Time PCR System  using KAPA SYBR4 cells/well in 12-well plates. The cells were transfected with TOPFlash reporter (0.5 μg) using Lipofectamine 2000 . Cells pretreated with specific inhibitors or overexpressing Myc-S6K1  or stably expressing S6K1 shRNA were treated with 20 mM LiCl for the indicated time points. Reporter activity was measured using the Dual-Luciferase Reporter Assay System  according to the manufacturer’s instructions.Cells were cultured at an equal density of 5 × 10Cells were equally seeded on each plate, and samples were obtained by trypsinizing one plate from each group daily for counting the number of cells. Each sample was counted using Luna-II™ automated cell counter according to manufacturer’s instructions.HT-29 and DLD-1 cells were seeded in 6-well plates at 80% confluency. After 24 h, wounds were created by scraping with pipette tip, and the cells separated from the plate were removed by washing twice with medium. Migration images were taken at 0, 1, and 2 days after wound creation. t-test and results with * p < 0.05, ** p < 0.01, *** p < 0.001 were considered significant (n.s.: no significance). Statistical analysis was performed using GraphPad Prism (version 7.0) . Statistical significance was calculated by performing Student’s


The microstructure, phase identification, surface bonding and composition of the nanoparticles were characterized by SEM, XRD, FT-IR, and Raman spectroscopy, respectively. The hardness of the coating was assessed by the pencil hardness method. The abrasion resistance of the coating was tested by a sand washing machine. The corrosion resistance of the coating was assessed using salt spray, a long-period immersion test, potentiodynamic polarization curves and electrochemical impedance spectra. With the addition of a small amount of nanoparticles, the dispersion of nanoparticles in the epoxy resin was good. When the content of nano-Al2O3 particles was equal to 1.5 wt%, the particles in the epoxy exhibited the best dispersion and stability. However, the GO and Al2O3@GO nanofillers in the epoxy resin exhibited poor dispersion and stability. The hardness, abrasion and corrosion resistance of the composite coatings were improved with the addition of a small amount of nanoparticles, but the performance began to decline after exceeding a certain content range of the nanoparticles. A relatively good abrasion resistance for the coatings was obtained when the content of Al2O3, GO and Al2O3@GO after modification was 1.5 wt%, 0.2 wt% and 0.4 wt%, respectively. The corrosion resistance of the coatings doped with nano-Al2O3 particles was better than that of the coatings incorporating GO nanosheets and Al2O3@GO hybrids. The corrosion mechanism of the composite coatings in 3.5 wt% NaCl solution was addressed and studied.Nano-Al 2O3 particles and graphene oxide (GO) nanosheets were modified by 3-aminopropyltriethoxysilane (KH550), and then dispersed in epoxy resin, and finally modified-Al2O3/epoxy, modified-GO/epoxy and modified-Al2O3@GO/epoxy composite coatings were prepared on steel sheets by the scraping stick method.Nano-Al Microcracks and holes are generated in the epoxy coating because of the influence of the high crosslinking density of the network. Corrosion media such as H2O, O2, and Cl− ions will permeate the epoxy coatings through some minor defects, which will reduce the service term of the coatings in the corrosive environment. Nanoparticles provide a new approach for modifying epoxy resin to obtain good mechanical properties and abrasion and corrosion resistance of the epoxy coatings. Nanoparticles have many unique and excellent properties. The insulation oxide ceramic nanoparticles, such as TiO2, SiO2, Al2O3, and ZrO2, acted as fillers for epoxy resin, which can improve the properties of the epoxy resin. Among the numerous oxide ceramic nanoparticles, the low-cost nano-alumina (nano-Al2O3) is well-known for its excellent mechanical properties, high thermal stability, super electrical insulation properties, and high surface area, which can be acted as a candidate reinforcement filler.2 However, due to the small size and high specific surface area, nano-Al2O3 particles are easy to agglomerate, resulting in its poor dispersion, which could influence the properties and performance of the composite coatings.3 Salimi et al.4 prepared Al2O3/tetraglycidyl-4,4′-diaminodiphenylmethane nanocomposites by adding nano-Al2O3 particles modified by aminopropyltriethoxysilaneandglycidylpropyltrimethoxysilane into the epoxy. The dispersion of nanoparticles in the resin influenced curing exothermic and fracture morphology of the composites, the dispersibility of nanoparticles modified by silane was good.5 The mechanical properties and corrosion resistance of the epoxy coatings can be significantly enhanced with the addition of nano-Al2O3.6–8In the field of anticorrosion, water-borne epoxy resin plays an important role due to its excellent mechanical and chemical properties.9 These functional groups make GO have good wetting and high surface activity, and can improve the compatibility between GO and epoxy resin10. Recently, the corrosion and tribology properties of graphene-based epoxy coating have attracted tremendous attention, due to its super physical shielding and high lubricity.11–13 Cui et al.14 reported an eco-friendly water-borne epoxy coating by embedding GO nanosheets, and found that inclusion of well-dispersed GO-polymerized polydopamine nanosheets led to the remarkable improvement in the corrosion resistance of water-borne epoxy coating. However, during the preparation process for the coatings, the dispersibility of GO nanosheets is one of the key factors due to the agglomeration of GO nanosheets with their high specific area, and nanoscale internal van der Waals forces, which limits the improvement in the abrasion and corrosion resistance of the epoxy coatings.15,16 The charged GO nanosheets17 or a new fangled cationic dopamine-reduced GO18 nanosheets that can stably disperse in the water-based epoxy. The highly parallel GO nanosheets tremendously improve the physical barrier effect of the coatings and prolong the penetration path of the corrosive medium. Li et al.19 added the epoxy resin into the ethanol solution containing sodium polystyrene sulfonate modified GO by the ultrasonic, stirring and volatile solvent methods to realize the uniform filling of GO. Pathak et al.20 used the phase transfer method to realize the uniform dispersion of GO in epoxy resin. Li et al.21 studied the properties of silane-coupling-agent-modified GO composite epoxy coatings, and showed that modified GO remarkably enhanced the mechanical properties of epoxy coatings.Graphene oxide (GO) has a two-dimensional lamellar structure and large specific surface area, which contains many active functional groups, such as hydroxyl group (–OH), carboxyl group (–COOH), epoxy group (–CH(O)CH–).2O3) hybrids have the physical barrier effect of GO and reinforcement of nano-Al2O3, resulting a hybrid property of the improvement in the mechanical properties-corrosion resistance of the composite coatings.22 Nano-Al2O3 particles are acted as a secondary filler to isolate the GO nanosheets. At the same time, nano-Al2O3 particles can weaken the van der Waals force of GO nanosheets and improve the dispersibility of GO nanosheets in epoxy resin23. Osman et al.24 found that the settling of nano-Al2O3 particles on the graphene surface not only inhibited the electron transfer but also eliminated the agglomerations of graphene. However, the excess addition of nanoparticles in epoxy resin also influenced the dispersibility. Therefore, the dispersion and dosage of nano-Al2O3 and GO nanosheets in epoxy resin are the key factors to improve the performance of the composite coatings, so it is of great significance to solve the dispersion of nano-Al2O3 and GO nanosheets in epoxy resin. Furthermore, seven different hybrid ratios of RGO/Al2O3 were dispersed by ultrasonication into the epoxy matrix, and found that the synergy of RGO/Al2O3 at 6 : 4 enhanced the thermal, insulation and mechanical properties of epoxy resin. Zhou et al.25 studied ZrO2 nanoparticles were controllably anchored on the reduced-GO (rGO) nanosheets via an environmentally friendly single-step hydrothermal reaction and then incorporated into epoxy coatings to simultaneously improve the wear resistance and anti-corrosion performance of the coatings, The addition of 0.5 wt% ZrO2@rGO nanohybrids to the epoxy coating significantly improved the adhesion strength, impact resistance and hardness, and the average friction coefficient decreased by ∼42% and the wear resistance improved by ∼57.7%. The as-received ZrO2@rGO hybrid in epoxy coatings could effectively prevent electrolyte penetration, and ZrO2 particles played a synergistic role with rGO nanosheets in alleviating corrosion. ZrO2 particles uniformly grew on the surface of rGO nanosheets to form nanohybrids with good dispersity. Yu et al.26 reported that GO–Al2O3 hybrids were fabricated using GO as a precursor, then anchoring Al2O3 on GO sheets with 3-aminopropyltriethoxysilane. GO–Al2O3 hybrids not only achieved an homogeneous dispersion and compatibility in epoxy resin, but also exhibited an obvious superiority in reinforcing the anti-corrosion performance of epoxy coatings. However, the concentration of GO, Al2O3, and GO–Al2O3 hybrids were not optimized and deeply studied.Graphene oxide–alumina  were purchased from Suzhou Tanfeng Graphene Tech Co. Ltd, China. The following chemicals were purchased from Shanghai Nalle New Material Technology Co., the epoxy resin , curing agent  and 37% hydrochloric acid (HCl). Silane coupling agents 3-aminopropyltriethoxysilane (KH-550) was provided by Chengdu Branch of Chinese academy of sciences. N,N-Dimethylformamide (DMF) was provided by Chengdu Long March Chemical Reagent Factory. The deionized water (D.I. water) was produced by a water purification machine .Nano-Al2.22O3 or mGO (modified Al2O3 or modified GO), nano-Al2O3 or GO with 2 g weight was added into 50 mL anhydrous ethanol and then ultrasonically dispersed for 30 min. Meanwhile, the hydrolysis pretreatment osilane coupling agent was carried out. 20 mL KH-550, 72 mL anhydrous ethanol and 8 mL D.I. water were evenly mixed for 30 min and then the pH of the mixture solution was adjusted to 3.0 with 1.0 M HCl. Then, the mixture solution was treated while stirring using a mechanical stirrer at 60 °C for 4 h. After centrifugation process at a rotate speed of 1200 rpm for three times, every time was 10 min, and then washed with anhydrous alcohol and D.I. water, finally dried in a vacuum oven at 80 °C for 24 h to obtain functionalized m-Al2O3 or mGO nanoparticles, as shown in 2O3 nanoparticles with 0.1 g weight was dispersed in 250 mL N,N-dimethylformamide (DMF) to form a homogeneous suspension by mechanical stirring. GO with 0.4 g weight was added into the suspension via ultrasonication for 1 h. The mixture solution was reacted and stirred for 5 h at 85 °C, and the as-received m-Al2O3@GO hybrids were washed with anhydrous alcohol and D.I. water for three times in the centrifuge at a high rotate speed of 1200 r min−1, then dried at 60 °C in a vacuum oven for 24 h. These mixture resins filled with nanoparticles were divided into a glass bottle to observe the dispersity and stability of nanoparticles in expoy resin.The synthesis procedure for modified nanoparticles is shown in 2.32O3 and mGO-Al2O3 hybrids by mechanical agitation at a rotate speed of 1300 r min−1 for 15 min to form a homogeneous dispersion system, and then taken into a constant temperature water bath at 60 °C until bubbles were removed in epoxy resin. Meanwhile, the size of the tinplate sheets was 70 × 17 × 3 mm3. Before adding the curing agent to the epoxy resin according to the volume ratio of 2 : 1, and then stirred slowly and uniformly for 10 min. The composite coatings were scraped on the tinplate sheets by a scraping rod. The thickness of the coating was controlled at around 70 ± 1 μm and dried naturally . Contact angles of the coatings were recorded using a goniometer . The liquid was distilled water, 5 μL in volume, which were dropped on the surface of the samples, in order to measure the contact angle. The final contact angle was determined three times at different places on the surface of the samples.The microstructure and morphology of nanoparticles were obtained by scanning electron microscopy . The crystal phases of the coating and particles were performed by X-ray power diffractometer  with Cu Kα radiation working in the Bragg–Brentano (θ–2θ) geometry utilizing a para-focusing geometry to increase intensity and angular resolution in the angle range of 6–78°. The chemical bonds and functional groups for modified nanoparticles were tested by the Fourier-transform infrared spectroscopy  spectra in the range from 400 to 4000 cm2.5The hardness of the coating was obtained by pencil hardness test, in accordance with the standard GB/T 6739-2006, and a pencil with a certain hardness was applied to the coatings. The scratches, indicated by the mark of the hard pencil, did not cause damage to the surface of the coatings.2.6−3 and a particle density of 3.96 g cm−3. The sand washing height and a diameter of sand washing pipe were 1.0 m and 50 mm, respectively. The surface of the coating was subjected to sand punching strength, and the angle between the sample and the flow direction of sand was 45°. The fine sands were collected in sand bucket, and then reused. The flushing volume of sand for each time was 2 L.The abrasion resistance of the coating was assessed by a sand shaker, and the schematic diagram of sand shaker is shown in 2.72. Then, the samples were placed in the 3.5 wt% NaCl aqueous solution open to the air, simulating the seawater environment. According to the national Standard GB/T 1771-2007 ‘color paint and varnish-neutral salt fog performance test’, the composite coatings were placed in the salt spray test box . The corrosion conditions were: 5 wt% NaCl aqueous solution, chamber temperature of 35 °C, saturation temperature of 47 °C, pH of 6.5–7.2 and spray volume of 1–2 mL/80 cm2 h−1. The angle between the tested surface of the sample and the vertical direction was fixed at 20°. The exposed area of the composite coatings was around 120 × 50 mm2.The opposite surface of the sample was glued with waterproof adhesive glue, and the exposed area was 10 × 30 mm2 exposed to 3.5 wt% NaCl aqueous solution. The electrochemical workstation (CHE 660E) was used to test the open circuit potential (OCP) and polarization curves of the samples. A standard three-compartment cell was used with an Ag/AgCl 3 M KCl electrode and a Pt electrode as a reference and counter electrodes, respectively. The potentiodynamic current–potential curves were recorded at a sweep rate of 20 mV min−1. Before the polarization test, the electrochemical impedance spectroscopic (EIS) measurements were carried out at the measured steady-state OCP value of the corresponding working electrode in the frequency range of 10−2 to 105 Hz. All the experiments were conducted at room temperature. All impedance measurements were made in Faraday cages to minimize external disturbances and the experimental data were fitted by ZsimDemo software. The corrosion rate (CR) of the composite coatings can be calculated by the following M is the atomic mass of the metal (MFe = 55.845), Icorr is the corrosion current density (A cm−2), ρ is the density of the corroding material (ρ = 7.85 × 103 kg m−3), and Z is the number of electrons transferred per metal atom (Z = 2).The samples used as a working electrode was bonded with a conductive wire by a conductive adhesive and then covered with acrylic resin leaving a square surface area of 1 cm33.12O3 and GO nanosheets are shown in 2O3 presents nearly spherical particles with significant agglomerations  and mGO (100) was 7.39 nm and 7.75 nm, respectively. The increase in interlayer space indicated that silane molecules and alkyl chains were successfully grafted onto the surface of GO nanosheets. Moreover, the diffraction peaks of nano-Al2O3 were also revealed in the XRD pattern of mGO@Al2O3 hybrids , 1515 cm−1 (N–H), and 1130 cm−1 (Si–O–Si),32,33 which were attributed to KH550, and importantly, the peak at 655 cm−1 belonged to Si–O–Al bonding,34,35 depicted in 2O3 particles were functionalized and modified by KH550 via chemical grafting. In −1 (–OH), 1629 cm−1 (C–OH) and 1074 cm−1 (C–O–C) were clearly observed,36 indicating that GO contained a large number of oxygen-containing hydrophilic groups and adsorbed water. The disappearance of the C–OH bonding vibration peak at 1074 cm−1 after KH550 surface modification was attributed to the reaction of KH550 with hydroxyl groups and the formation of Si–O–C bonding. Two new absorption peaks appeared at 2924 cm−1 and 2851 cm−1, which were the stretching vibration peaks on the methyl group and methylene group on the KH550.37 It was indicated that KH550 was grafted onto the chemical bonding of GO. For m-Al2O3@GO hybrids  was absent, implying the reaction between the epoxide of GO and amino group of the KH550.35,38,39 The results showed a referential value for the effect of the interaction between m-Al2O3 nanoparticles and GO nanosheets to some extent.FT-IR spectra of the nanoparticles are shown in  hybrids , the bro2-carbon atoms, D-band corresponds to the presence of vacancies or distortions in the carbon rings, and the 2D-band is valuable to predict the number of graphene layers.40,41 After surface modification, G- and D-bands were shifted to lower wavenumbers of 1561 cm−1 and 1325 cm−1, respectively. The appearance of D-band in Raman spectrum for GO nanosheets was due to the introduction of oxygen moieties between GO nanosheets in which most of the sp2 bonds were transformed into sp3 bonds. The destruction of structural tube symmetry of GO was due to the decrease in the size of the sp2 graphitic domains upon oxidation, leaving a disorganized and amorphous structure.42,43 The intensity ratio between D- and G-bands (ID/IG) is used to determine the structural integrity of graphene and its degree of disordering.44 After the surface functionalization of GO, the intensity ratio of mGO was 0.769, which was significantly lower than that of virgin GO (0.952). The vacancies and defects in the structure of GO were reduced after surface modification with KH550, and these vacancies and defects were caused by the reduction of oxygen functional groups.24 The intensity ratio of 2D-band and D-band is used to predict the number of GO layer. If the intensity ratio of 2D-band and D-band is high, the number of GO layer is few. Therefore, the number of mGO layer is much less than that of virgin GO, because of no evidence of 2D-band in Raman spectrum.45 This result was proved by SEM image in 3.22O3, some particles gradually appear on the surface of the coating. As shown in 2O3/epoxy coating may be caused by poor compatibility with epoxy resin. A large number of m-Al2O3 particles are observed on the surface of 4.5 wt% m-Al2O3/epoxy coating  and (b)),2O3@GO particles were added, the hydrides on the surface of the coating are evenly distributed and occasionally large particles existed  and The OM images of the top-surface of the coatings are shown in  coating . On the  coating , a small existed  It is ob3.32O3 nanoparticles, the contact angle decreases and the hydrophilicity improves. The addition of mGO and hybrid will also further reduce the contact angle of the coating, and the effect of these two additions is much more obvious than that of m-Al2O3, 1.0 wt% m-Al2O3@GO/epoxy has the smallest contact angle and the largest hydrophilicity, with a contact angle of 57.65°. Al2O3 itself is hydrophobic,59 which verifies that Al2O3 particles have been successfully modified by KH550 from hydrophobic to hydrophilic. FT-IR shows that GO contained a large number of oxygen-containing hydrophilic groups and adsorbed water, as a result, the hydrophilicity of GO and Al2O3 is improved after hybridization.The contact angles of the composite coatings are shown in 3.42O3 was still an uniform suspension. The m-Al2O3 nanoparticles were well dispersed in epoxy resin and kept stable for a long time. As the holding time increased ,2O3@GO/epoxy also began to delaminate. On the 55th day, almost all hybrid nanofillers were precipitated at the bottom of the bottle and the dispersion of 1.0 wt% m-Al2O3@GO/epoxy was better ,46ncreased , the del3.52O3, 0.4 wt% mGO and 0.4 wt% m-Al2O3@GO hybrids increased by two grades. The main reason was that when the nanoparticles were added to the epoxy resin, m-Al2O3 nanoparticles formed a tight grid structure with the epoxy, so that the composite coatings have high hardness. In addition, the modified nanoparticles were surrounded by a large number of epoxy functional groups, which can bond with the amine curing agent added to the coating and enhance the bonding force between the molecules. However, as the addition amount of m-Al2O3@GO hybrid was increased to 1.0 wt%, the hardness of the coating was reduced to 3H due to the agglomeration of nanoparticles, resulting in the weakness in the adhesion between the epoxy matrix and nanoparticles.47,48 The adhesion of the coatings on the substrates was assessed by cross-cut testing with 3M tape, as shown in Table S1.2O3@GO hybrids was less than that of m-Al2O3 for the same hardness values of the coating.3.62O3 particles were added to the epoxy, the abrasion resistance of the composite coatings was better than that of the neat epoxy coating, with sand flushing for several times and small fracture area can be observed. When the addition amount of m-Al2O3 particle was 1.5 wt%, the abrasion resistance of the coating was greatly improved , due to no protection by the red glue for the coating. In Fig. S6(b),2O3@GO/epoxy coatings. In Fig. S6(c),2O3@GO/epoxy coating had poor corrosion resistance, but the corrosion resistance of the coatings with less 0.6 wt% m-Al2O3@GO was relatively good. The pitting corrosion (see the red circled area) was observed on the surface of the coatings. The formation of the corrosion rust involved several redox reactions 49The coatings were immsered in 3.5 wt% NaCl solution for a long-term period .2O3 particle was better than that of the coating with low-content mGO nanosheets and m-Al2O3@GO hybrids. For the composite coatings filled with mGO nanosheets and m-Al2O3@GO hybrids, the coatings with less content of fillers showed relatively good corrosion resistance, due to the good dispersibility and stability of the low-content nanofillers in the epoxy resin.According to the above discussion, the corrosion resistance of the coating with high-content m-Al3.7.22O3/epoxy composite coatings had better corrosion resistance, in comparison to the other coatings  the epoxy resin could be regarded as a physical barrier coating, (2) Al2O3 had anti-corrosion performance inherently, (3) the nanoparticles provided an extra barrier layer to preeminently obstruct micropores for electrolyte permeation, which prevented the underlying metal from corrosion attack, and (4) the well-dispersed m-Al2O3 nanoparticles in epoxy resin prevented corrosion due to a relatively high aspect ratio, which could enhance corrosion resistance of the composite coatings. The mGO nanosheets were added and stacked in the epoxy matrix, which was used as an effective barrier to capillary pores diffusion, and to prolong the tortuosity of the diffusion pathway of O2 molecules to penetrate the coatings  nanosheets were successfully modified by KH550 and m-Al2O3@GO hybrids were synthesized using a facile approach. Then, the epoxy resin was filled with m-Al2O3, mGO and m-Al2O3@GO hybrids. The dispersibility and stability of particles in the epoxy resin had an influence on the hardness, abrasion and corrosion resistance of the composite coatings. The main conclusions were summarized below:Nano-Al2O3 was equal to 1.5 wt%, the m-Al2O3 nanoparticles in the epoxy exhibited the best dispersibility and stability. However, the dispersibility and stability of mGO and m-Al2O3@GO nanofillers by mechanical stirring in the epoxy resin were not good.(1) With the addition of small amount of nanoparticles, the dispersion of nanoparticles in the epoxy resin was good. When the content of m-Al(2) The hardness of the composite coatings increased with increasing particle content and decreased after exceeding a certain content of the particles, which was related to the dispersibility and stability of the particles in the epoxy resin.2O3/epoxy composite coatings had better abrasion resistance, compared with the mGO/epoxy and m-Al2O3@GO/epoxy composite coatings with different degrees of the damaged area. The composite coatings incorporated with 1.5 wt% m-Al2O3, 0.2 wt% mGO and 1.0 wt% m-Al2O3@GO had relatively good abrasion resistance.(3) The m-Al2O3/epoxy coatings was better than the mGO/epoxy and m-Al2O3@GO/epoxy coatings. The corrosion resistance of the composite coatings was sorted in the order of 1.5 wt% m-Al2O3/epoxy coating > 0.6 wt% m-Al2O3@GO/epoxy coating > 0.4 wt% mGO/epoxy coating > the neat epoxy coating.(4) For the different composite coatings, the coatings with less content of nanofillers showed relatively better corrosion resistance. And the corrosion resistance of the m-AlThere are no conflicts to declare.RA-012-D2RA04223A-s001