Three-dimensional (3D) printing, alternatively known as additive manufacturing, is a transformative technology enabling precise, customized, and efficient manufacturing of components with complex structures. It revolutionizes traditional processes, allowing rapid prototyping, cost-effective production, and intricate designs. The 3D printed graphene-based materials combine graphene’s exceptional properties with additive manufacturing’s versatility, offering precise control over intricate structures with enhanced functionalities. To gain comprehensive insights into the development of 3D printed graphene and graphene/polymer composites, this review delves into their intricate fabrication methods, unique structural attributes, and multifaceted applications across various domains. Recent advances in printable materials, apparatus characteristics, and printed structures of typical 3D printing techniques for graphene and graphene/polymer composites are addressed, including extrusion methods (direct ink writing and fused deposition modeling), photopolymerization strategies  and powder-based techniques. Multifunctional applications in energy storage, physical sensor, stretchable conductor, electromagnetic interference shielding and wave absorption, as well as bio-applications are highlighted. Despite significant advancements in 3D printed graphene and its polymer composites, innovative studies are still necessary to fully unlock their inherent capabilities. In recent years, the exploration of innovative materials and advanced manufacturing techniques has become pivotal in addressing the growing demand for multifunctional materials with tailored properties across a multitude of applications. Among these materials, graphene has garnered exceptional attention due to its remarkable mechanical strength, high thermal and electrical conductivity, and impressive surface area . GraphenThree-dimensional 3D) printing, or additive manufacturing, is a process of creating 3D objects by adding materials layer by layer based on a digital design or model, which allows for the creation of complex and customized shapes directly from a digital file without the need for traditional subtractive methods such as cutting or drilling D printin,5. The aOver the last decade, the field of 3D printing has witnessed a surge in research focused on graphene-based materials, resulting in a diverse array of hierarchical structures with remarkable properties. The extrusion-based, photopolymerization-based, and powder-based 3D printing techniques have been confirmed to be effective in fabricating graphene and graphene/polymer composites ,10,11,12Typically, the process of 3D printing involves several routines: (i) preparing printable materials; (ii) designing the structures using 3D modeling software, such as computer-aided design software and a 3D scanner; (iii) slicing the 3D model into 2D layers; and (iv) printing materials layer by layer ,14. SeveThe extrusion techniques (DIW and FDM) involve the deposition of material in a controlled manner through a nozzle or extrusion system, building objects layer by layer via extruding and depositing materials in a semi-liquid or molten state. Extrusion techniques offer versatility in terms of material compatibility, as they can accommodate a range of materials such as thermoplastics , thermosDIW enables the precise deposition of functional inks to create complex and customizable 3D structures. In DIW, a viscous ink is dispensed through a nozzle or similar extrusion system and is directly written onto a substrate or previously printed layers . Inks fo−1 σysh≥Though GO dispersions are widely applied as inks for DIW, not all of them are suitable. Several techniques have been developed for the optimization of GO inks, including strengthening GO networks by (i) increasing strengths of inks with a higher concentration and larger lateral size, (ii) gelling induced by inter-sheet crosslinking, and (iii) adding rheology modifying additive . By care4 and 100 Pa, respectively, which are suitable for DIW-based 3D printing.The mechanical properties and viscosity of colloidal GO dispersions are related to the colloid volume concentration. Generally, four different states of GO suspensions were observed with different GO concentrations: viscoelastic liquid, transition region, viscoelastic soft solid, and liquid crystalline gel, as shown in 2+ acts as crosslinkers of GO sheets, turning the GO sol into a viscous hydrogel, as shown in 2+ ions lead to changes in the rheological behaviors of the GO solution, increasing its viscosity and storage modulus. This allows the hydrogel to maintain stability and flow, making it suitable for use in DIW for 3D printing , G, G88], G0.01 wt% . The per0.01 wt% ,90,91,92The apparatus of SLA mainly consists of a laser source that generate UV lasers for polymerization of photopolymers, a lens and an XY coordination mirror system to control the moving of the UV laser, a build platform that can move up and down to accumulate photopolymerized composites layer by layer, as shown in Different from the extrusion-based techniques discussed before, SLA-printed objects generally show relatively layerless and continuous build. SLA is a form of vat photopolymerization, where a liquid photopolymer resin is selectively cured by a focused UV laser to solidify it into the desired shape. This process results in objects with smooth and homogeneous surfaces c,d, lackDLP utilizes a digital micromirror device (DMD) and photopolymer resins to create three-dimensional objects, which shares similarities with SLA in terms of using photopolymerization to solidify liquid resin . HoweverIn addition to the DLP printing system, the graphene/photopolymer is critical to the final DLP printed objects . The genBy taking advantage of the solubility of GO in ethanol as well as the miscibility of ethanol and acrylic resins, a concentrated GO liquid crystal ethanol solution with sufficient viscosity was prepared by Tilve-Martinez and coworkers for DLP printing, as shown in Sharing similar printing mechanisms with SLA (despite the different UV light sources), DLP printing selectively cure an entire layer of liquid resin on the latest cured layer. As a result, the transitions between layers are generally smoother and less pronounced compared to methods that build objects through the incremental deposition of material, such as DIW and FDM. The continuous and smooth surfaces of DLP-printed objects are clearly shown in The SLS is a typical powder-based additive manufacturing process that is suitable for the fabrication of graphene/polymer composites. Selective laser melting is another commonly used powder-based 3D printing, while it is more applicable for metallic materials . In the As described, the SLS is based on the manufacturing of powders; therefore, the quality of the building powders directly influences the final 3D printed objects. Spherical polyvinylidene fluoride (PVDF) powders (average diameter of 200 nm) and GO powders (average diameter of 0.5–3 μm and average thickness of 0.55–1.2 nm) were blended in ethanol which can disperse GO sheets but do not dissolve PVDF powders . After u2 laser on carbon-containing material, such as polyimide (PI), cloth, paper, and food, in ambient atmosphere [The SLS was integrated with the laser-induced graphene (LIG) process to print neat graphene objects. The LIG is a 3D porous material prepared by direct lase writing with a COmosphere ,124. A Lmosphere . On the The morphology of graphene nanocomposites fabricated through SLS is highly diverse, intricately influenced by factors such as powder characteristics, sintering parameters, and post-processing treatments. However, similarly, within the SLS procedure, where layers of material are meticulously fused by a precision laser, the graphene sheet-coated polymer powders coalesce to unveil a distinctive segregated distribution . As showWhile each section of 3D printing techniques briefly touched upon the post-processing of 3D printed graphene and graphene/polymer composites, this section aims to offer a more comprehensive exploration of the pivotal post-processing steps. It is recognized that diverse 3D printing methods demand distinct post-processing approaches to optimize material properties and end-product quality. Recognizing the unique attributes of each 3D printing technique, this section will delve into the tailored post-processing strategies for each method.2/rGO aerogel lattices via post-electrodeposition of MnO2 nanosheets, thereby conferring exceptional supercapacitive performances upon the printed lattice [Upon completion of the DIW printing process, the removal of residual solvents from the printed objects becomes imperative. To achieve this, three primary strategies have been developed, namely freeze drying, supercritical fluid drying, and ambient-temperature drying, all aiming to preserve structural integrity while facilitating effective drying . Freeze  lattice .The characteristic “staircase effect” in FDM printed materials is readily discernible, presenting a distinct layer-by-layer morphology. To address this, thermal annealing emerges as a critical intervention, facilitating an advanced level of fusion and bonding amidst the sequentially deposited layers . This trComparable post-processing steps are requisite subsequent to SLA and DLP processes which both employ the solidification of liquid resin layer by layer via UV exposure. Upon the completion of printing, the object typically undergoes immersion in a solvent bath, which is essential to eradicate surplus uncured resin . This stUpon the completion of the SLS process, the printed object is carefully extracted from the surrounding loose powder, followed by elimination of surplus powders by either precise brushing or controlled blowing . As discBased on the above review on 3D printing techniques for graphene and graphene/polymer composite, comparisons of the above-discussed five methods are shown in Supercapacitors, also known as ultracapacitors or electrochemical capacitors, are a type of energy storage device that store and release electrical energy quickly and efficiently. They operate on the basis of electrostatic charge storage within the electrochemical double layer at the electrode–electrolyte interface, offering high power density, a long cycle life, and quick charging. This makes them suitable for various applications such as electric vehicles, renewable energy systems, and consumer electronics . TherefoThe 3D printed graphene structures show superiority over traditional thick-film electrodes in terms of supercapacity, as shown in Many efforts have been made to optimize the supercapacitance of 3D printed graphene ,140,141.3 solution at a potential of 1.9 V versus saturated calomel electrode [2 at an ultrahigh power density of 164.5 mW/cm2. A graphene aerogel microlattice fabricated by Ca2+ crosslinking-assisted DIW exhibited wrinkled porous structure and coherent conductive network, showing gravimetric capacitance of 213 F/g at 0.5 A/g and 183 F/g at 100 A/g, as well as a retention over 90% after 50,000 cycles [2 was developed by DIW-based printing of GO ink followed by freeze drying, thermal annealing and electrodeposition of MnO2 nanosheets, as shown in 2 (182.2 mg/cm2) on the 3D printed graphene aerogel electrode yielded high areal capacitance , excellent conductivity, and efficient ion diffusion. Through the symmetric arrangement of two 4 mm-thick electrodes the resultant device exhibited a notable energy density of 1.56 mW h/cm2. Moreover, this electrode, comprising a 3D-printed MnO2-deposited graphene aerogel, achieved exceptional capacitance values normalized to area, mass, and volume simultaneously, overcoming the typical trade-offs associated with most electrode materials.The supercapacitive properties of 3D printed graphene aerogels were further enhanced by surface functionalization of graphene sheets using electrochemical oxidation in 0.5 M KNOlectrode . With an0 cycles . A 3D-pr2), high energy density (4.17 μW h/cm2) and power density (0.22 mW/cm2). When printed on a flexible substrate, flexible supercapacitors are fabricated [In-plane microsupercapacitors with a compact size are a type of microscale energy storage device designed to be integrated into electronic circuits or systems. The addition of spacers of carbon sphere and a self-sacrificing template of ethyl cellulose in graphene inks enabled the 3D printing process of a graphene-based interdigital-structured microsupercapacitors, as shown in bricated ,148.3D printing for graphene-based batteries represents an innovative approach to fabricate advanced energy storage devices that harness the unique properties of graphene . Graphen2 batteries [2 batteries [2 when considering the total battery area. This configuration yielded initial charge and discharge capacities of 117 and 91 mA h/g, respectively, while maintaining commendable cycling stability.3D printing allows for intricate and complex battery designs, which can be tailored to specific applications. 3D printed GO framework with thermal shock synthesized nanoparticles for Li-COatteries  and holyatteries  are typiatteries . 3D prinatteries . An all-atteries . A metic2 plasma treatment. The 3D porous anode structure with high surface areas decreased the local current density and inhibited the growth of sodium dendrites. The N doping through controllable plasma treatment reduced the nucleation potential and promoted uniform sodium ion flux, which make additional contributions to the uniform deposition of sodium metal  into the 3D printed graphene microlattices was also beneficial to reduce the local current density and enhance the uniform nucleation and deposition of sodium metals [Another contribution of the 3D porous graphene-based architecture is its ability to inhibit the growth of metallic dendrites during charge and discharge cycling. As shown in m metals . Similarm metals .Working as substantial intermediates for collecting external mechanical signals, flexible strain sensors are regarded as indispensable components in flexible integrated electronic systems. Graphene-based strain sensors is an emerging and promising application that leverages the exceptional electrical properties of graphene to create highly sensitive and customizable sensors for measuring mechanical deformation or strain. The 3D printing technique has recently been used to fabricate flexible sensors due to its high accuracy, simplicity, and rapid prototyping ,158. GO Hybrid filler are applied to construct the conductive networks for optimization of 3D printed graphene-based strain sensor. FDM-printed high-performance flexible strain sensors using CNT and graphene nanoplatelet-filled TPU composite filaments showed a high sensitivity , a wide detectable strain range (0–250%) and good stability . In addiThe printing resolution of additive manufacturing plays an important role in determining performances of 3D printed strain sensors. A higher printing resolution results in more functional units exist per unit area, which can accurately sense deformations of objects (such as robot hand) in the form of resistance changes. Chen et al.  developeThe combination of artificial intelligence and robotics has become one of the fast-growing research fields and is considered to have a profound impact on human soci-ety. The development of robotic skin capable of perceiving external pressure environments, or the pressure sensing, is highly desirable for intelligent robots. Besides, pressure sensors find extensive applications in various industries, including automotive, aerospace, medi-cal, industrial automation, consumer electronics, and environmental monitoring. Based on the electrically conductive nature of graphene sheets, the graphene or gra-phene/polymer composite pressure sensors are generally piezoresistive, which directly convert the external pressure to an electrical signal  ,171,172.Taking advantages of the flexibility of structural design by 3D printing, different conductive networks  can be designed, showing different pressure sensing capacities ,176. HigLaser-induced graphene with a layered alignment and porous structure is suitable for pressure sensing. With careful adjustment of the laser power, a laser-induced graphene on-skin sensor for on-body monitoring, robotic hand control, and embedded machine learning for signal classification was designed . 3D prin−1. Extrusion-printed CNT/graphene lines using hybrid inks with different ratios of CNT and graphene exhibited temperature-dependent resistance with a significantly large negative temperature coefficient of resistance, showing an averaged resistance drop (per unit temperature) of −3.5 Ω/°C [−1, and it can be integrated using all-printed all-in-one configuration composed of a asymmetric microsupercapacitor and the temperature sensor [3D printing techniques also show great significance in temperature sensing ,181,182.3.5 Ω/°C . A 3D pr3.5 Ω/°C . A tandee sensor .−1 (0–60 kPa) and a high temperature sensitivity of 30.8 μV/K [By constructing complex geometries of graphene/polymer composite by 3D printing, flexible pressure– or strain–temperature dual parameter sensors utilizing independent piezoresistive and thermoelectric sensing mechanisms, respectively, can be achieved. A graphene-coated carbon nanofiber (CNF)/TPU architectures endowed the sensor with a high pressure sensitivity of 0.14 kPa0.8 μV/K . This se0.8 μV/K .Flexible temperature sensors often grapple with the challenge of sensing material deformation, leading to complications in maintaining sensing accuracy and reliability due to external strain-induced resistance fluctuations. Wang and colleagues addressed this issue by utilizing DIW to create graphene/PDMS composites with intricate geometries such as grids, triangles, and hexagons for strain-insensitive temperature sensing . the celThe addition of electrically conductive graphene sheets into a polymer matrix is a common strategy to make the intrinsically insulating polymer conductive . To achi3, an electrical conductivity of 278 S/m and a super-compressibility up to 90% compressive strain. DIW printing of electrically conductive graphene aerogel monolith using large-size GO sheets precursors enhanced the compressibility (up to 80% compressive strain), electrical conductivity (41.1 S/m), specific strength (10.7 × 103 N m/kg) and lowered density (12.8 mg/cm3) [2+ gelled GO/CNT hybrid inks were used for DIW printing of aerogels, followed by pre-buckled reduction, to produce highly stretchable graphene/CNT hybrid aerogels [With rational design, DIW printed graphene aerogel lattices can serve as flexible conductors, either stretchable or compressible. Zhu et al.  develope mg/cm3) . Ca2+ geaerogels . The higaerogels .The incorporation of elastomer matrices significantly enhances the mechanical robust, flexibility and stability of printed objects. Robust, flexible and conductive multilayer 3D printed graphene/polycaprolactone composites containing 10 wt% graphene were fabricated by the DIW process . Highly 3D graphene monoliths with controlled macrostructures can be fabricated through DIW using GO inks , but genCoating graphene nanosheets or graphene-based hybrid conductive materials on 3D-printed porous structure is another effective approach to combine carbon nanomaterial with 3D porous polymer substrates and thus to manufacture high-performance stretchable conductive materials. The eco-friendly and easy-moving PLA was firstly 3D printed to form a designed structure with spacing between neighboring skeletons being 200 μm, followed by backfilling PDMS with the assistant of vacuum and subsequent PLA etching . The porOwing to the fast development of modern electronics, communications, and industries, the spatial environment in earth is constantly exposed to electromagnetic radiations which are hazardous to our health and safe operation of electronic devices. To minimize the negative effects of electromagnetic radiations, electromagnetic interference (EMI) shielding and wave absorbing materials are developed. Mechanisms behind the EMI shielding of a material mainly includes reflection , absorption attenuation , and multiple reflection (negligible when absorption-induced shielding effectiveness (SE) is larger than 10 dB) of electromagnetic waves ,202. The2/g) in the frequency range of 8.2–12.4 GHz. A local enrichment strategy was developed to fabricate graphene/PLA nanocomposites for FDM printing by precisely manipulating the selective distribution of fillers, obtaining segregated graphene sheets in the printed object [The FDM method is widely used for the fabrication of 3D printed EMI shielding materials . The stud object . The resd object . The cord object .3O4 functionalized graphene/polymer nanocomposites achieved a high electrical conductivity of ~580 S/m and an EMI SE of over 50 dB in the X-band [3, the DIW printed and frozen dried scaffold achieved an ultrahigh normalized surface specific SE of up to 19,270 dB cm2/g.In situ reduction of GO in PDMS solutions followed by viscosity adjustment and DIW printing provided highly stretchable and conductive graphene/PDMS composite lattices, as shown in e X-band . When ine X-band . The DIWe X-band . The arce X-band . AttribuWave absorption, also known as electromagnetic wave absorption, is a process by which specific materials or structures absorb and dissipate electromagnetic energy in the form of heat. Different from EMI shielding which is about creating a barrier to prevent electromagnetic waves from entering or escaping a device, wave absorption focuses on dissipating electromagnetic energy as heat to reduce the intensity of the waves and reduce the reflection of incident radiations . Wave abThanks to the flexibility and effectiveness of 3D printing in structural design, multilayer rGO/PLA absorbers were designed with a gradient index of characteristic impedance by adjusting the rGO content and the geometric parameters of the unit cell, with the seven-layer material giving an absorption above 90% in a broad bandwidth of 4.5–40 GHz . A funct0.35Zn0.3Fe2.35O4/PMMA as the matching layer and CIG/PMMA as the absorption layer via multi-material DLP, achieving a minimum reflection loss of −46.1 dB at 4.7 GHz and an effective absorption bandwidth of 3.5 GHz (4.15–5.35 and 14.7–17.0 GHz) [2-graphene/PLA double-layer absorber with effectively improved impedance matching using FDM technology [There are many other explorations of 3D printing in wave absorption. For example, the DLP printed CIG/poly (methyl methacrylate) (PMMA) nanocomposites with various compositions presented a minimum reflection loss of −54.4 dB at a thickness of 2.1 mm, along with effective absorption bandwidth of 3.41 GHz . Zuo et 7.0 GHz) . The bal7.0 GHz) . Ye et achnology . A minimco-glycolide were also demonstrated to be able to support human mesenchymal stem cell adhesion, viability, proliferation, and neurogenic differentiation with significant upregulation of glial and neuronal genes [The biocompatible graphene is capable of supporting stem cell growth and osteogenic differentiation as well as adsorbing dexamethasone and β-glycerolphosphate (osteogenic inducers) via π–π stacking, making it beneficial bone tissue engineering . Additival genes . Scaffolal genes . These sS. aureus on the surface of scaffold under direct current [The 3D printed graphene/polymer scaffold are also found to be promising in antibacterial applications. A polymer scaffold that was reinforced with silver nanoparticle decorated GO sheets realized sustained release of Ag ions from the scaffold, demonstrating an antibacterial performance  . The GO  current .Graphene/polymer composite with well-defined structures fabricated by 3D printing have also been explored for many other bio-applications. For example, 3D printed graphene/PMMA composites with customized shape and good antimicrobial, stiffness and strength properties were proposed to be suitable for dental repair . An autoIn this review, we provide an overview of recent advances in 3D printed graphene and graphene/polymer composites. The commonly applied additive manufacturing techniques, including extrusion-based methods (DIW and FDM), photopolymerization strategies (SLS and DLP), and the powder-based technique (SLS), for graphene and its polymer-based composites are comprehensively reviewed. The unique combination of graphene’s exceptional properties and the design flexibility of 3D printing opens up exciting opportunities for the development of advanced materials with a wide range of functionalities. The ability to tailor the structural characteristics and properties of the 3D printed architectures enables the creation of custom-designed components for various fields, including energy storage, sensor, stretchable conductor, EMI shielding and bio-applications. Even though 3D printed graphene and graphene/polymer composites have been demonstrated to be promising in multifunctional applications, there still are important issues as follows to be addressed to unlock their full potential.Owing to the layer-by-layer deposition fabrication process, the interlayer strength of 3D printed materials remains a critical aspect affecting their mechanical integrity and overall performance. Inadequate interlayer adhesion can lead to delamination, reduced strength, and compromised functionality, limiting the practical application of 3D printed graphene composites. More attentions on enhancing the interfacial strength of 3D printed objects are essential. Several strategies can be employed to enhance interlayer adhesion. First, optimizing print parameters, including temperature, layer height, and printing speed, is essential to achieve uniform and strong interlayer bonding. Ensuring consistent printing conditions reduces the risk of weak interfaces and delamination. Second, surface modification of graphene or polymer materials can improve their compatibility, promoting better interfacial bonding between adjacent layers. Third, the incorporation of interlayer bonding agents or adhesion promoters during the printing process can strengthen interlayer connections and enhance the overall adhesion. Additionally, post-processing techniques, such as annealing or surface treatments, are also promise in promoting interlayer bonding and improving the mechanical properties of 3D printed composites. A comprehensive understanding of the factors influencing interlayer strength and the implementation of these strategies will advance the field of 3D printed graphene composites.As for 3D printed graphene-based materials, the diversity in materials, structures and functionalities is not fully explored yet. The 3D printing technique, with its ability to integrate multifunctional materials and complex structures seamlessly, can enable the creation of all-in-one customized devices with unprecedented capabilities. By incorporating different types functional materials, microscopic and macroscopic structures, the 3D printed composites can be endowed with integrated functionalities, meeting several or all specific requirements in a particular application. For example, with rational design of the material and structure, multifunctional sensors capable of detection and discrimination of different physical stimuli  are possibly be integrated by 3D printing techniques. The integration and de-coupling of multifunctional materials provides opportunities for graphene-based materials to be applied in complex situations.In the realm of 3D printed graphene and graphene/polymer composites, the advantages stemming from the flexibility in structural and material design have only scratched the surface of their true potential. While researchers have made significant strides in leveraging this unique capability, there are vast unexplored opportunities awaiting discovery. The ability to tailor the structural characteristics and properties of these composites provides unmatched versatility, allowing the creation of custom-designed components for a wide range of applications. However, the full extent of this design freedom remains untapped. As 3D printing technologies evolve and our understanding of graphene’s behavior improves, we can expect groundbreaking advancements in the field. By exploring innovative material combinations, advanced additive manufacturing techniques, and optimized print parameters, we will witness the emergence of novel multifunctional materials with exceptional mechanical, electrical, thermal, and even biological properties.


Idiopathic pulmonary fibrosis (IPF) is a lung disease that is both chronic and progressive and is characterized by glycolysis. However, glycolysis’s function and its clinical significance in IPF are still not well understood.We accessed the Gene Expression Omnibus database to retrieve mRNA expression information for lung tissue and other samples. We identified genes associated with glycolysis that had differential expression levels between IPF and controls. In this work, we conducted a comprehensive bioinformatic analysis to systematically examine the glycolysis-associated genes with differential expression and subsequently investigated the possible prognostic significance of these genes. Additionally, the expression profiles of the associated prognostic genes were further investigated via quantitative real-time polymerase chain reaction in our cohort. In this investigation, we found that the expression of 16 genes involved in glycolysis was differentially expressed. Among them, 12 were upregulated and 4 were downregulated. We found that 3 glycolysis-related genes  might serve as hub genes for anticipating patient prognosis. The data from these genes were used to generate the prognostic models. The findings confirmed that high-risk IPF patients recorded a shorter overall survival relative to low-risk patients. This prognostic model yielded 1-, 2-, and 3-year survival rates of 0.666, 0.651, and 0.717, correspondingly, based on the area under the curve of the survival-dependent receiver operating characteristic. The GSE27957 and GSE70866 cohorts validated these findings, indicating the model has a good predictive performance. All 3 glycolysis-associated genes were validated to be expressed in our cohort. Finally, we used mRNA levels from 3 genes to produce a nomogram to quantitatively predict the prognosis of IPF individuals. As possible indicators for the prognosis of IPF, the glycolysis-related genes stanniocalcin 2, transketolase like 1, and artemin were shown to be promising candidate markers. The incidence of IPF is low in young people and more common in the elderly. The median survival duration following diagnosis ranges between 2 and 4 years.,3 Due to the development and use of antifibrotic drugs (pirfenidone or nintedanib) in recent years, the prognosis of IPF individuals has improved to some degree. However, IPF is still incurable, in part because its etiology is complex and the mechanism is unclear. Therefore, further comprehension of the molecular basis that underlies IPF is crucial for improving the prognosis, early screening, and diagnosis of IPF patients.Idiopathic pulmonary fibrosis (IPF) has been recognized as a chronic, irreversible, progressive interstitial lung disorder with undetermined etiology and poor clinical prognosis.,7 Fibrosis may develop as a consequence of lung macrophage activation and subsequent inflammation, which might in turn induce a proteolytic phenotype in macrophages and reduce lung elastin levels. Active macrophages rely heavily on glycolysis and high lactate generation to swiftly supply the energy needed to sustain the inflammatory response. Aerobic glycolysis is commonly referred to as the Warburg effect where cells can still carry out glycolysis under the condition of sufficient oxygen, convert glucose into pyruvate, and process it into lactic acid, consequently promoting cell growth and proliferation. Researchers have indicated that in pulmonary fibrosis, aerobic glycolysis is considerably enhanced. It has been shown that inhibiting aerobic glycolysis may alleviate bleomycin-mediated lung fibrosis and reduce the rate of TGF-β-induced collagen formation.,11 The results of Xie et al’s study also showed that lung fibroblasts had enhanced glycolysis and upregulation of glycolysis enzymes in myofibroblasts that have been differentiated in vitro by TGF-β. The findings of Kottmann’s research indicated the presence of lactic acid accumulation in patients with IPF. The above research data suggest that the process of aerobic glycolysis of lung tissue might cause fibrosis by activating TGF- β and other fibrogenic factors through the survival and accumulation of lactic acid. Up to now, the pathogenicity of glycolysis in IPF is still worthy of further study. A better comprehension of the role glycolysis performs in the IPF has led to the accumulation of more data supporting the treatment of patients with the disease. The results of the investigation by Yin et al indicated that when bleomycin-mediated pulmonary fibrosis mice were treated with HK2 inhibitors, the expression of fibrogenic genes was reduced and lung function remained stable. Pulmonary fibrosis is associated with glycolysis, however, the specific molecular mechanisms that underly glycolysis in the lungs are not well characterized. Therefore, the study on the systematic function of glycolysis-associated genes in IPF is warranted to elucidate the function of glucose metabolism in IPF and find novel targets for IPF treatment.Pulmonary fibrosis is also a metabolic-related disease, and the changes in glucose metabolism perform an integral function in the formation of pulmonary fibrosis.This research aimed to examine the functions of glycolysis in IPF and assess the prospective clinical value of differentially expressed glycolysis-associated genes for prognostic stratifications. In particular, the Gene Expression Omnibus (GEO) database was searched for data on mRNA expression and clinicopathological characteristics. We conducted bioinformatics analysis to identify genes involved in glycolysis with differential expression between individuals with IPF and healthy controls and extensively analyzed the possible biochemical processes and functions of these genes. Lastly, we combined glycolysis-related gene expression data with clinical data to examine the possible influence of these genes on prognosis.http://www.broad.mit.edu/gsea/msigdb/) is a repository of annotated gene sets for Gene Set Enrichment Analysis. Four glycolysis-associated gene sets were extracted, containing KEGG GLYCOLYSIS GLUCONEOGENESIS, BIOCARTA GLYCOLYSIS PATHWAY, HALLMARK GLYCOLYSIS, and REACTOME GLYCOLYSIS . An aggregate of 255 glycolysis-associated genes was retrieved from MSigDB, which offers gene annotations in great depth. We retrieved the Next Generation Sequencing data of 103 IPF and 103 control lung tissues with corresponding clinical data from GSE 150910. Information on patients in the GSE150910 dataset is displayed in Table https://bioconductor.org/packages/limma/) was utilized to execute the negative binomial distribution technique with the absolute value of log2(fold change) > 1.0 and false discovery rate < 0.05 to find differentially expressed glycolysis-associated genes between IPF and control lung tissues. The most common recursive feature-removal algorithm, support vector machine-recursive feature elimination (SVM-RFE), was utilized to choose the features for removal. The differentially expressed genes were used to complete SVM-RFE analysis, which could be used to screen the optimal feature genes in the GSE150910 dataset.The molecular signature dataset  for anticipating the PPI network. In the meantime, the R software  was employed to visualize the PPI network.The glycolysis-associated genes showing differential expression were imported into the Search Tool for the Retrieval of Interacting Genes .where β signifies the coefficient value and Exp indicates the levels of gene expression. Subsequently, the median approximated risk scores were applied to classify IPF patients into low- and high-risk groups (categories). The “prcomp” function included in the “stats” R package was applied to conduct the principal component analysis of the 3-gene signature. Next, the log-rank test was conducted to contrast the prognostic results of the 2 cohorts. We investigated the predictive power of the aforementioned model via receiver operating characteristic (ROC) analysis utilizing the SurvivalROC R package ( and GSE70866(176 IPF patient samples with matching prognosis) datasets were employed to verify the estimated findings and assess the model’s prognostic performance. The information on the genechip is shown in Table The GSE27957(45 IPF patient samples with matching prognosis)Ethical approval was obtained from the ethics committee of the First Affiliated Hospital of Chengdu Medical College (IRB ID:2021CYFYIRB-BA-32–01). Each subject signed the informed consent. The TRIzol reagent  was used for the extraction of total RNA from PBMCs. After the determination of total RNA purity and concentration, reverse transcription of the total RNA into cDNA was done utilizing the PrimeScript RT reagent kit . SYBR Green Premix Ex Taq II was utilized to execute the qRT-PCR. The PCR parameters were as illustrated below: 95°C for 30 seconds, ensued by 40 cycles of 95°C for 5 seconds, and 60°C for 30 seconds for each particular primer. Lastly, the 2−△△Ct method was applied to derive the relative mRNA expression profiles of the 3 genes. Table P values < .05 denoted the significance criterion.We likewise utilized real-time PCR to verify the expression of the 3 prognostic genes in the research cohort. Beginning July 2021 to September 2022, we acquired 15 peripheral blood mononuclear cells (PBMCs) from IPF patients and 15 PBMCs from healthy individuals at the First Affiliated Hospital of Chengdu Medical College in Sichuan, China. The eligible subjects for this research included adult patients clinically diagnosed with IPF as per the European Respiratory Society guidelines/American Thoracic Society/(2011).To elucidate a quantitative method to predict an IPF patient’s probability of glycolysis-related genes, we constructed a nomogram based on the “rms” R program.Genes involved in glycolysis were systematically analyzed to determine their potential predictive performance and key roles in IPF. The investigation was carried out exactly as planned, as illustrated in Figure To find glycolysis-associated genes that have a significant link to overall survival (OS), a univariate analysis that was based on hub glycolysis-related genes was conducted. As per the hazard ratio forest map, there was a strong link between the expression of 8 glycolysis-associated genes and the IPF patients’ prognoses Fig. A. The 8 The glycolysis-related prediction model was built using the 3-hub glycolysis-associated genes discovered in the preceding analysis. Each IPF patient’s risk score was determined as illustrated below:Risk score = (−0.561*ExpSTC2) + (−0.247*ExpTKTL1) + (0.177*ExpARTN).Subsequently, we performed a survival analysis to evaluate how well this model predicted patient outcomes. By using the median of their risk scores, 75 individuals with IPF were categorized into high- or low-risk categories. The principal component analysis illustrated that patients exposed to varying risks were grouped into 2 cohorts . These findings suggested that this predictive model predicated on glycolysis-related genes exhibited good specificity and sensitivity. Additionally, we validated the expression of the 3 key genes in the study sample. As projected, STC2 and TKTL1 were significantly decreased in IPF patients, while ARTN was increased  in mammals exhibit a widespread expression in various tissues.,30 On the human chromosome, the STC2 gene is found at position 5q35.1. In both humans and mice, STC2 is made up of 4 exons that span 13kb of DNA. Upregulation of STC2 in N2a mice neuroblastoma cell lines after being subjected to thapsigargin- and tunicamycin-stimulated estrogen receptor stress was the first piece of evidence for STC2’s cytoprotective role, presented by Thinakaran et al. An upregulation of STC2 was also observed following hypoxia and oxidative stress mediated by H2O2. When STC2 was overexpressed, it protected cells against thapsigargin-stimulated cell death in both HeLa and N2a cell lines. Kim et al demonstrated that the overexpression of STC2 triggered the pERK1/2 and pAKT signaling pathways, protecting cells from oxidative stress-triggered cell injury. In our study, bioinformatics analysis and qRT-PCR results suggested that STC2 was lowly expressed in IPF. Cox univariate regression analysis showed that the hazard ratio value of STC2 was 0.570, suggesting that STC2 was a protective factor. We hypothesized that after STC2 expression was reduced, the protective effect on alveolar epithelial cells was lost, and alveolar epithelial cells were over-repaired after repeated injury and converted to fibroblasts, which finally led to the formation of pulmonary fibrosis. It is still worthwhile to investigate STC2’s function in pulmonary fibrosis.STC, which is a hormone containing a glycosylated peptide, initially identified in a bony fish was previously demonstrated as having an integral function in phosphate and calcium homeostasis. In the pentose phosphate pathway, TKTL1 is a rate-limiting enzyme that is abundantly expressed in epithelial-derived normal and tumor cells. Consequently, TKTL1 performs an integral function in the survival, growth, proliferation, migration, and differentiation of cells, and participates in the incidence and advancement of numerous human illnesses.–37 At present, extensive research points to a strong association between TKTL1 and malignant growth. Although TKTL1 is overexpressed in non-small cell lung cancer, it is unrelated to patient outcomes in terms of OS, disease-free survival, or tumor node metastasis stages. In this study, TKTL1 is a protective factor in IPF. Verified by qRT-PCR, TKTL1 is lowly expressed in PMBCs of IPF patients. There is currently no investigation conducted on the link between TKTL1 and IPF. Whether TKTL1 participates in the formation and development of IPF is worthy of further study.TKTL1 is one of the 3 members of the human TKT gene family . Ceyhan et al showed that in chronic pancreatitis, GFRalpha3 and ARTN exhibited a substantial overexpression and were sited in neural ganglia, Schwann cells, and smooth muscle cells of arteries. Also, the elevated ARTN mRNA levels were found to correlate with hypertrophy, neural density, perineural inflammatory cell infiltration, inflammation, and pain severity. In addition, both ARTN expression and neural alteration exhibited a positive link to the severity of fibrosis. Pancreatic fibrosis and pulmonary fibrosis have some similarities in pathological features and mechanisms, which are caused by repeated damage of epithelial cells, leading to the formation of scars in tissue. The results of bioinformatics analysis in this research illustrated that ARTN was increasingly expressed in IPF tissues, and ARTN had a negative correlation with the prognosis of IPF. Combined with the previous literature, we speculate that ARTN might be implicated in the inflammatory cell infiltration in pulmonary fibrosis and promote the formation of IPF. Song et al illustrated that ARTN may enhance lung cancer cell migration and invasion, which might be related to hypoxia-induced ARTN-promoting epithelial-mesenchymal transition (EMT) through AKT signaling. In addition to being related to tumor invasion and metastasis, EMT is also involved in typical wound healing and performs a function in the excessive repair of tissues in IPF.,43 Therefore, we believe that ARTN might be implicated in the EMT process of IPF. This research verified the high expression of ARTN in PBMCs of IPF patients by qRT-PCR, but further research is needed to improve the mechanism.ARTN is a well-recognized affiliate of the glial cell line-derived neurotrophic factor ligand family . Its signal transduction mainly regulates intracellular signal transduction by combining with the co-receptor GFR α 3-RET to regulate intracellular signal transduction, thus affecting neural system development and cell homeostasis. also developed a nomogram using the 3 inflammation-associated genes in IPF. We employ several bioinformatics analytic approaches that are similar to some of the procedures used by Lu et al.Following that, a risk model for predicting IPF prognosis was developed utilizing multiple stepwise Cox regression analyses of 3 glycolysis-related genes, which was then validated using the GSE27957 and GSE70866 cohorts. The ROC curve analysis illustrated that the 3 glycolysis-related genes signature was fitted for forecasting dismal prognosis in IPF patients. Furthermore, the 1, 2, and 3-year OS rates were intuitively predicted using a nomogram. This predictive model centered on these 3 glycolysis-related genes is both monetarily cost-effective and clinically advantageous. Lu et alThis prediction method, however, was discovered to have some limitations. Firstly, the model was built using the GEO dataset; thus, it needed to be validated in a clinical patient population. Also, the number of patients included in our study was small. Further expansion of the sample size is required to validate this model in the future. Secondly, because this was retrospective research, more prospective studies are required to confirm the findings. Lastly, functional studies are required to additionally uncover the possible mechanism of glycolysis-related genes that might be important in IPF.Ultimately, following a thorough bioinformatics analysis of the GEO database, our investigation discovered 3 IPF prognostic glycolysis-related genes. The findings provided new insights into the functions performed by glycolysis in IPF pathogenesis, perhaps paving the way for the creation of prognostic indicators.Conceptualization: Yu Li, Yaju Deng, Jie He.Data curation: Yu Li, Jie He.Formal analysis: Yaju Deng, Jie He.Investigation: Jie He.Methodology: Jie He.Project administration: Yaju Deng, Jie He.Resources: Yu Li, Yaju Deng, Jie He.Software: Yu Li, Jie He.Supervision: Jie He.Validation: Yu Li, Jie He.Writing – original draft: Yu Li, Yaju Deng.Writing – review & editing: Yu Li, Yaju Deng.


A detailed theoretical analysis of the Fourier transform of quasiparticle interference images reveals that the induced SOC consists of a valley-Zeeman (λvZ ≈ 2 meV) and a Rashba (λR ≈ 15 meV) term, one order of magnitude larger than what theory predicts, but in excellent agreement with earlier transport experiments. The validity of our analysis is confirmed by measurements on a 30 degree twist angle heterostructure that exhibits no backscattering, as expected from symmetry considerations. Our results demonstrate a viable strategy to determine SOC quantitatively by imaging quasiparticle interference.Inducing and controlling spin-orbit coupling (SOC) in graphene is key to create topological states of matter, and for the realization of spintronic devices. Placing graphene onto a transition metal dichalcogenide is currently the most successful strategy to achieve this goal, but there is no consensus as to the nature and the magnitude of the induced SOC. Here, we show that the presence of backscattering in graphene-on-WSe Graphene has many intriguing electronic properties. One of note is the absence of backscattering of electrons confined to a single valley. Spin-orbit interactions can allow backscattering, and here, Sun et al. use this spin-orbit coupling dependence of backscattering to measure the strength of the spin-orbit interaction in a graphene/tungsten selenide heterostructure. Key in this context is the notion of backscattering—the ability of electrons to precisely reverse their direction of propagation as a result of elastic collisions—because how electrons backscatter is deeply related to the structure of the electronic wavefunctions and to the symmetries present in the system3. For electrons described by scalar wavefunctions in conventional metals, for instance, time reversal symmetry causes constructive interference that enhances backscattering and suppresses conductivity, resulting in the well-known phenomenon of weak-localization4. In the presence of SOC—for which the spinorial structure of wavefunctions needs to be considered—time reversal symmetry instead results in destructive interference that suppresses (rather than enhancing) backscattering, leading to weak antilocalization3.Phase coherent propagation of electrons in solids is a basic quantum mechanical process that determines the low-temperature transport properties of metallic conductors8. The difference is rooted in the two-component, pseudo-spinorial wavefunction of Dirac electrons, which originates from the two inequivalent atoms forming the graphene unit cell10. The Hamiltonian of pseudo-spinorial Dirac fermions in graphene possesses an additional symmetry—so-called chiral symmetry9—which ensures that electrons with opposite momenta have orthogonal pseudospins  measurements, and succeed in relating our observation to the properties of SOC. Images acquired on heterostructures with different twist angles between the SLG and WSe2 lattices give fully consistent results as to the type and magnitude of the SOC imprinted by the WSe2 layer onto SLG. We find that both Rashba and valley-Zeeman (vZ) terms are present, with a magnitude varying between 7 and 16 meV for the Rashba contribution, and between 1 and 3 meV for the vZ. These values are in perfect agreement with the results of earlier magnetotransport studies15, and establish that the strength of the Rashba term is one order of magnitude larger than that predicted by existing ab initio calculations19. We further find that no quasiparticle interference is observed for SLG-on-WSe2 when the twist angle is 30°, in agreement with theoretical expectations20, because for this angle the strength of the vZ coupling vanishes by symmetry and backscattering is only present when both vZ and Rashba are non-zero. Besides settling pending issues about the type and strength of SOC in SLG-on-TMD, our work demonstrates a conceptually new approach to probe spin-orbit coupling quantitatively in 2D materials.Here, we establish the occurrence of backscattering of Dirac electrons in single-layer graphene (SLG)-on-WSe21, the ability to controllably generate SOC artificially, while preserving the basic electronic properties of Dirac electrons, is of interest in different contexts24. A route towards this goal consists in imprinting SOC by placing graphene on semiconducting TMD substrates such as WSe2, which combine a very large SOC strength 26 with a large band gap around the graphene Fermi level. A variety of transport experiments34 exhibiting unambiguous signatures of a strong SOC in graphene show that this route is successful. However, most experiments detect SOC exclusively through a fast spin relaxation33, much faster than that expected in pristine graphene. This unfortunately provides only limited information, since a fast spin relaxation can be understood in terms of spatially random spin-dependent scattering, and does not require that the SOC imprinted by the TMD substrate corresponds to a modification of the graphene band structure. Therefore, most reported studies which analyse fast spin relaxation times, neither give conclusive evidence for the presence of spin-splitting in the dispersion relation of Dirac electrons, nor do they give unambiguous indications as to the type of SOC imprinted in graphene. Only a few experiments indicate that the SOC imprinted by the TMD substrate onto graphene does induce a modification to the band structure15, and attribute the resulting spin-splitting near the Dirac point to two distinct SOC terms. These are a Rashba term (which wants the electron spin to point in the graphene plane) and a valley-Zeeman term (which forces the electron spin to point in the direction perpendicular to the graphene plane). The experiments consistently suggest a strength of the Rashba and vZ terms of ≈15 meV and ≈2 meV, respectively (for graphene on WS2). When compared to ab initio calculations19, these results are only partially in agreement: whereas the observed strength of the vZ term indeed corresponds to the expected one, no calculation has so far found a Rashba term as strong as the one extracted from the experimental data. Whether this deviation originates from the assumptions in the theoretical modeling or from problems with the interpretation of the experiments remains to be established. At this stage, it is crucial to push experiments further, by pursuing alternative strategies to probe SOC in graphene on TMDs, capable of providing quantitative information on the strength of the different SOC terms.Given the very weak (≈50 μeV) SOC naturally present in graphene36. The Fourier transform (FT) of QPI images gives a finite signal at qB in the QPI thus serves as a direct measure of the existence of backscattering.In the presence of elastic scattering, the wave of an incoming electron with momentum mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minimmentclass2pt{minim2 stacked with selected twist angles between 1° and 30°  and the Dirac point, whose shallow depression near −197 mV is consistent with previous studies37. Detecting backscattering by QPI relies on large area high resolution 2 lattice peaks, the corresponding moiré peaks, and their linear combinations. The six ring patterns near the K and K’ points correspond to intervalley scattering sketched in Fig. 39.We measured the QPI images on different heterostructures consisting of mechanically exfoliated SLG and few-layer WSeintravalley scattering contributions around the Γ-point, which are illustrated in full detail in Fig. k = 0. Such a behavior has been observed earlier in bilayer graphene40—where it has been shown to be due to backscattering—but is not expected to occur in pristine SLG, since backscattering is in principle forbidden39. The observed feature is clearly reminiscent of the manifestation of intravalley backscattering introduced in Fig. Figure qB of the ring at which intravalley scattering peaks, decreases with reducing energy from 30 meV to −20 meV. We quantify the precise dependence of qB on energy by analyzing angle-averaged line cuts  of Dirac electrons (as demonstrated by the linear dispersion relation with the correct Fermi velocity). We conclude that in contrast to pristine SLG, where no backscattering is observed, backscattering of Dirac electrons is present in SLG-on-WSe2.The evolution of the ring around the Γ-point with electron energy Fig. a as well2, we proceed with a fully detailed theoretical analysis of the data. To model this, we add to the usual single-valley Dirac Hamiltonian the Rashba term HR = λR(τzσxsy − σysx) and the vZ term HvZ = λvZτzsz, where σ refers to the sublattice pseudospin, τ to the K/s to the physical spin. HvZ acts like a valley-dependent magnetic field, splitting spin up and down in different directions in the K and 2. By symmetry, λvZ must vanish in the heterostructure with a 30° twist angle between graphene and the WSe2 layer19, which has important experimental consequences (see below). The Rashba term couples spin and pseudospin, leading to spin split bands with opposite pseudospin and spin windings. When both vZ and Rashba are present, the pseudospin vectors are mixed with the spin degree of freedom, and these spin/pseudospin vectors are no longer opposite for opposite momenta, so that nothing prevents backscattering. Importantly—since both the vZ and Rashba coupling s have to be non-zero for the argument to hold—backscattering is expected to disappear for a 30° twist angle between SLG and WSe2, when symmetry imposes that λvZ = 0.Having established the presence of intravalley backscattering of Dirac electrons in SLG-on-WSe2 with SOC, in the limit of small disorder. We have also modelled the effect of a mass gap, by adding to the Hamiltonian a spin-independent term of the type HΔ = mσz, because such a term can also induce backscattering in SLG. However, in contrast to SOC, whose presence for SLG-on-TMD is established, there is no earlier experimental indication that a sizable mass gap is present in these heterostructures: a sizable mass gap should lead to an insulating behavior at low-temperature, which has never been reported in transport experiments .Using the model Hamiltonian with the vZ and Rashba terms, we have calculated the theoretical QPI patterns . For the vZ term, the strength is approximately constant upon varying twist angle . The result of the experiments is shown in Fig. 2 and validates our conclusions as to the strength of the vZ and of the Rashba terms.These results demonstrate that the analysis of QPI images in SLG-on-WSegle Fig. c; for thgle Fig. d. The er2 this technique allows a full characterization of the SOC terms present, which is key to establish a firm experimental understanding of the spin-orbit coupling imprinted in graphene by proximity effect. We anticipate that the analysis of similar QPI imaging measurements on other material systems, in conjunction with detailed theoretical modeling, will also enable to obtain detailed information about spin-orbit coupling.Determining what type of SOC is present in the band structure of an electronic system and estimating the magnitude quantitatively are notoriously complex issues. On 2D materials, only a few techniques have been employed so far to extract this information, all based on the study of low-temperature magnetotransport as a function of gate voltage in devices with sufficient high mobility. The use of scanning tunneling spectroscopy to perform quasiparticle interference imaging to achieve this same goal demonstrates a strategy that had not been pursued earlier. It relies on a specific phenomenon—in the present case, whether backscattering occurs or not—that can be probed by careful STM measurements and modeled in detail theoretically. As we have shown here, on SLG-on-WSe44. An exfoliated single-layer graphene and a few-layer thin WSe2 crystal were picked up with a defined twist angle by a propylene carbonate (PC) film coated on polydimethylsiloxane (PDMS) stamps. This stack was subsequently released on a 90 nm SiO2/Si substrate  at 4.5 Kelvin using an electrochemically etched tungsten tip. The tips were further conditioned in situ on Au(111). The bias voltage is applied to the sample, with 0 V corresponding to the Fermi level. To acquire reproducible high-resolution scanning tunneling microscope (STM) images and 13. Second, the devices were annealed at 350 °C for 180 min in a H2/Ar atmosphere before transferring them into the STM chamber. Third, the devices were annealed once more at 350 °C for an hour in ultra-high vacuum. Topographic STM images were acquired in constant-current mode. The Vrms = 2 mV at a frequency f = 429 Hz. The All STM data were acquired in ultra-high vacuum (base pressure < 10Supplementary InformationPeer Review File


The cytoplasmic accumulation of the nuclear protein transactive response DNA-binding protein 43 kDa (TDP-43) has been linked to the progression of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 secreted into the extracellular space has been suggested to contribute to the cell-to-cell spread of the cytoplasmic accumulation of TDP-43 throughout the brain; however, the underlying mechanisms remain unknown. We herein demonstrated that the secretion of TDP-43 was stimulated by the inhibition of the autophagy-lysosomal pathway driven by progranulin (PGRN), a causal protein of frontotemporal lobar degeneration. Among modulators of autophagy, only vacuolar-ATPase inhibitors, such as bafilomycin A1 (Baf), increased the levels of the full-length and cleaved forms of TDP-43 and the autophagosome marker LC3-II (microtubule-associated proteins 1A/1B light chain 3B) in extracellular vesicle fractions prepared from the culture media of HeLa, SH-SY5Y, or NSC-34 cells, whereas vacuolin-1, MG132, chloroquine, rapamycin, and serum starvation did not. The C-terminal fragment of TDP-43 was required for Baf-induced TDP-43 secretion. The Baf treatment induced the translocation of the aggregate-prone GFP-tagged C-terminal fragment of TDP-43 and mCherry-tagged LC3 to the plasma membrane. The Baf-induced secretion of TDP-43 was attenuated in autophagy-deficient ATG16L1 knockout HeLa cells. The knockdown of PGRN induced the secretion of cleaved TDP-43 in an autophagy-dependent manner in HeLa cells. The KO of PGRN in mouse embryonic fibroblasts increased the secretion of the cleaved forms of TDP-43 and LC3-II. The treatment inducing TDP-43 secretion increased the nuclear translocation of GFP-tagged transcription factor EB, a master regulator of the autophagy-lysosomal pathway in SH-SY5Y cells. These results suggest that the secretion of TDP-43 is promoted by dysregulation of the PGRN-driven autophagy-lysosomal pathway. The abnormal aggregation of the nuclear protein, transactive response DNA-binding protein 43 kDa (TDP-43) in the cytoplasm is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP) , 2. TDP-de novo pathology of cytoplasmic accumulation of TDP-43 in a mouse brain, which progressively spread throughout the brain in a time-dependent manner via the neuroanatomic connectome  haploinsufficiency because of a loss-of-function mutation in the PGRN gene causes FTLD-TDP , 33. Altvia EV. To elucidate the mechanisms underlying the release of TDP-43 into the extracellular space and the role of PGRN in this process, we examined the secretion of TDP-43 using different classes of modulators for autophagy. We used the v-ATPase inhibitors, Baf and concanamycin A (Con), the PIKfyve  inhibitor, vacuolin-1 (Vac), and the autophagosome–lysosome fusion inhibitor, chloroquine (CQ), the autophagy inducer, rapamycin (Rapa), and serum starvation (SS) as well as the knockdown of siRNA.In the present study, we aimed to examine whether the autophagy-lysosome pathway regulates extracellular release of TDP-43 A). HeLa cells expressing the fluorescence autophagy marker were treated with 100 nM Baf, an inhibitor of v-ATPase and autophagosome–lysosome fusion, 1 μM MG, an inhibitor of proteasome degradation and an inducer of autophagy, or 100 nM Vac, an inhibitor of PIKfyve and autophagosome–lysosome fusion, for 24 h . LC3-II-positive autophagosomes were identified as yellow puncta because of merged GFP and RFP signals and autolysosomes as red puncta because of the loss of the GFP signal under low pH (or acidic) conditions. Baf, MG, and Vac significantly increased yellow puncta, indicating a higher number of autophagosomes . On the other hand, the formation of autolysosomes was eliminated by Baf and Vac but not by MG . These results suggest that the increase in the number of autophagosomes induced by Baf and Vac was due to the suppression of autophagosome–lysosome fusion, whereas MG accelerated the formation of autophagosomes. Autophagic flux was assessed using HeLa cells stably expressing GFP-LC3-RFP-LC3ΔG (A and B). On the other hand, MG decreased the GFP/RFP ratio, indicating its facilitation of autophagic flux (C and D). The GFP/RFP ratio of Vac-treated cells was similar to that of vehicle-treated cells (C and D). Immunostaining of the phagophore marker WIPI2 (WD repeat domain phosphoinositide interacting 2) showed a significant increase in signal intensity with both Baf and MG but not with Vac (A and B). These results suggest that Baf and MG increased the formation of autophagosomes. The inhibition of lysosomal degradation using E64d and pepstatin A increased LC3-II levels in MG- or Vac-treated cells but not in Baf-treated cells (A–D), suggesting that Baf suppressed lysosomal degradation. Lysosomal acidity, assayed using AcidiFluor ORANGE staining, was elevated in cells treated with MG and Vac but was decreased by Baf (D and E). These results suggest the distinguishable effects of Baf and Vac on the inhibition of autolysosome formation.To elucidate the relationship between TDP-43 secretion and autophagy, we investigated whether stimulators of TDP-43 secretion, such as Baf, MG132 (MG), and Vac , 44, indFig. 1for 24 h , 46  for the collection of larger vesicles, followed by 110,000g (the P2 EV fraction) for the collection of smaller vesicles. The expression levels of cellular proteins were quantified using the lysates of adhered cells, and the loading volumes of samples from the P1/P2 EV fractions were normalized by protein levels in the cell lysates. Immunoblotting results showed that the cleaved form of TDP-43 was detected by the anti-C-terminal TDP-43 antibody, but not by the anti-N-terminal TDP-43 antibody, suggesting that full-length and C-terminal TDP-43 proteins were both released by the Baf treatment (F). To differentiate the cleaved variants of TDP-43 present in the cell, P1, and P2 fractions, we subjected each fraction to immunoprecipitation using the polyclonal rabbit anti-C-terminal TDP-43 antibody. Samples were then immunoblotted with the monoclonal mouse anti-TDP-43 antibody. Immunoprecipitation analysis revealed the presence of multiple variants of cleaved TDP-43 proteins in the cell fraction as well as the P1 and P2 EV fractions (A). Four bands  were detected in the cell fraction. Bands b and c were the predominant bands in the P1 EV fraction, whereas blurry band d was dominant in the P2 EV fraction (B). Densitometric data obtained from immunoblotting results on HeLa, SH-SY5Y neuroblastoma, and NSC-34 motor neuron–like cells showed that Baf, but not MG or Vac, significantly increased full-length and cleaved TDP-43 levels in the P1 and P2 EV fractions without affecting full-length TDP-43 expression levels in the cell fraction . The levels of the exosome marker TSG101 (tumor susceptibility gene 101 protein) in the P1 and P2 EV fractions were markedly increased by Baf but not by MG or Vac without any changes in the cellular expression levels . Baf, MG, and Vac increased the level of LC3-II, an autophagosome marker, in the cell fraction, whereas only Baf markedly increased LC3-II levels in the P1 and P2 EV fractions . These results suggest that the inhibition of autophagosome–lysosome fusion by Baf increased the secretion of full-length and C-terminal TDP-43 via vesicles associated with autophagosomes or exosomes.Cells release EV with the different size and intracellular origin, and the proteins enriched within these vesicles differ between the small and large sizes . To quanreatment F. To difractions A. Four bfraction B. DensitG and H, , A–C. ThA). Full-length TDP-43 levels in the cell fraction were higher in cells treated with Baf plus SS than in those treated with Baf only. On the other hand, cleaved TDP-43, TSG101, and LC3-II levels remained unchanged in the cell fractions of cells treated with Baf, Baf plus Rapa, and Baf plus SS . In the presence of Baf, the levels of full-length and cleaved TDP-43 and LC3-II were higher in the P1 EV fraction from cells treated with Rapa or SS compared with the untreated cells. The treatment with Baf plus SS increased TSG101 levels in the P1 EV fraction more than Baf alone or the Baf plus Rapa treatment . The levels of full-length and cleaved TDP-43, TSG101, and LC3-II in the P2 EV fraction were similar in cells treated with Baf, Baf plus Rapa, and Baf plus SS. SS significantly decreased cleaved TDP-43 levels in the P2 fraction . The autophagic flux assay using HeLa cells stably expressing GFP-LC3-RFP-LC3ΔG showed no change in the GFP/RFP ratio in response to the Rapa treatment and a decrease in response to SS, suggesting that SS, but not Rapa, facilitated autophagic flux (A and B). WIPI2 immunostaining revealed that both Rapa and SS, similar to Baf, increased WIPI2 signal intensity. Moreover, the combination of Baf plus Rapa or Baf plus SS induced a larger increase in WIPI2 signal intensity than the single treatment with Baf, Rapa, or SS (A and B). The inhibition of lysosomal degradation using E64d and pepstatin A increased LC3-II levels in cells treated with Rapa or SS, indicating that Rapa or SS did not suppress lysosomal degradation as effectively as Baf (A and B). These results suggest a positive correlation between the levels of secreted TDP-43 and the secretion of autophagy-associated vesicles triggered by the inhibition of autophagosome–lysosome fusion.Baf increased the formation of autophagosomes under the inhibition of autophagosome–lysosome fusion. To investigate the relationship between EV-mediated TDP-43 secretion and autophagy, we examined the effects of autophagy inducers on TDP-43 secretion, phagophore formation, autophagic flux, and lysosomal degradation in the presence or the absence of Baf. HeLa cells were treated with Baf, Rapa, SS, Baf plus Rapa, or Baf plus SS for 24 h and then subjected to assays A. Full-lreatment , B–E, P1fraction , B–E, P2gic flux , A and Ba, or SS , A and By as Baf , A and BA). Vesicles in the P1 EV fraction exhibited a wide range of sizes (approximately 50–1000 nm in diameter), whereas those in the P2 EV fraction were smaller (approximately 50–200 nm in diameter). Immunolabeling with the anti-C-terminal TDP-43 antibody confirmed the presence of both the full-length and cleaved forms of TDP-43. However, the TDP-43 fibril-like structures reported in a previous study  (C–E), indicating that EV derived from cells exposed to Baf shared some of the properties with previously reported EV , and the p62 band in the P2 EV fraction displayed a somewhat diffuse pattern   indicaterf.gov/) . Moreoverf.gov/) , ACTN4 arf.gov/) . The Baforted EV . The aut pattern . These rTARDBP) was silenced in HeLa cells using siRNA, and the Baf-induced secretion of autophagy-associated vesicles was then examined. As shown in A and B, full-length TDP-43 levels were significantly decreased in the cell, P1, and P2 fractions upon the knockdown of TARDBP (A and B). The knockdown of TARDBP significantly decreased cleaved TDP-43 levels in the cell and P1 EV fractions but not in the P2 EV fraction (C). TSG101 levels in the cell and EV fractions were not affected by the loss of TARDBP (D). The levels of LC3-II were significantly increased not only in the cell fraction, as previously reported (E). These results suggest that the loss of function of TDP-43 enhanced the cellular accumulation and the extracellular release of autophagy-associated vesicles induced by Baf.The accumulation of the nuclear protein TDP-43 in the cytoplasm of an affected region in FTLD-TDP or ALS patients causes not only the toxic gain of function of TDP-43 in the cytoplasm but also the loss of function of TDP-43 in the nucleus . Confocal live-cell images showed the prominent nuclear localization of TDP-43 amino acids 1 to 414 and TDP-43 amino acids 1 to 273. In contrast, TDP-43 amino acids 162 to 414 colocalized with mCherry-LC3 . Sequential centrifugation was conducted to identify which regions of TDP-43 were required for the Baf-induced secretion of TDP-43 . Equivalent levels of the TDP-43 amino acids 1 to 414, TDP-43 amino acids 1 to 273, and TDP-43 amino acids 162 to 414 proteins were detected in the cell fraction. The P1 EV fraction had lower levels of TDP-43 amino acids 1 to 273 than those of TDP-43 amino acids 1 to 414 and TDP-43 amino acids 162 to 414 (D). Only TDP-43 amino acids 162 to 414 were detected in the P2 EV fraction . These results suggest that the C-terminal 162 to 414 region of TDP-43 was required for the Baf-induced secretion of TDP-43.We examined the structural element of TDP-43 that was required for incorporation into EV using already characterized GFP-tagged full-length TDP-43 (amino acids 1–414), GFP-tagged N-terminal TDP-43 (amino acids 1–273), or GFP-tagged C-terminal TDP-43 (amino acids 162–414) . HeLa ceFig. 5A and C). In contrast, when cells were treated with Baf, TDP-43 amino acids 162 to 414 inclusions larger than 0.2 μm in diameter (B and D). These results suggest that the Baf treatment facilitated the fusion of autophagosomes incorporating TDP-43 with the plasma membrane.The Baf-induced incorporation of TDP-43 in autophagosomes with the plasma membrane was monitored using confocal live-cell imaging. Without the Baf treatment, TDP-43 amino acids 162 to 414 and mCherry-LC3 were detected near the plasma membrane but clearly distal , A and Cdiameter  increasediameter , B and DA and B). A deficiency of autophagosomes was confirmed by the loss of LC3-II production in ATG16L1 KO cells (C). Densitometric data revealed that ATG16L1 KO attenuated the incorporation of full-length TDP-43 in P1 vesicles and cleaved TDP-43 in P1 and P2 vesicles (D and E). On the other hand, ATG16L1 KO did not affect TSG101 levels in the cell or EV fraction (F). These results suggest that the formation of autophagosomes was necessary for the Baf-induced secretion of both the full-length and cleaved TDP-43 proteins.The loss of cellular ATG16L1 expression has been shown to suppress the formation of autophagosomes . TherefoKO cells C. Densitvesicles , D and Efraction F. These A–D). The formation of autolysosomes indicated by DALGreen (DALG) was also significantly suppressed by 100 nM Con, 100 μM CQ, or 100 nM Baf (A and B). These results suggest that both Con and CQ suppressed autophagosome–lysosome fusion, similar to Baf. On the other hand, the Con and CQ treatments increased WIPI2 signal intensity, which suggested that they promoted autophagosome formation, similar to Baf (A and B). In parallel, lysosomal acidity was suppressed by the treatment with 100 nM Con, 100 μM CQ, or 100 nM Baf as assessed by AcidiFluor ORANGE (C and D). The treatment with 100 nM Con increased the levels of both full-length and cleaved TDP-43 proteins in the P1 and P2 EV fractions, as did treatment with 100 nM Baf, but 10 or 100 μM CQ failed to induce the secretion of TDP-43 , which encodes a SNARE (soluble NSF attachment protein receptor) protein that regulates autophagosome–lysosome fusion  and lysosomal acidification indicated by the signal of AcidiFluor ORANGE (C and D), consistent with previous reports . Similarly, the knockdown of STX17 significantly increased LC3-II levels in the cell fraction but not in the P1 or P2 EV fraction (E and H). These results suggest that the suppression of autophagosome–lysosome fusion did not contribute to increases in the secretion of TDP-43.The roles of autophagosome–lysosome fusion in the secretion of TDP-43 were further examined in HeLa cells by the siRNA knockdown of the syntaxin 17 gene  in HeLa cells or NSC-34 motor neuron–like cells. The knockdown of GRN significantly decreased DALG (A and B) and AcidiFluor ORANGE (C and D) signal intensities, indicating the suppression of autolysosome formation and lysosomal acidification. The knockdown of GRN increased the levels of the cleaved TDP-43 protein in the P1 and P2 EV fractions, whereas full-length TDP-43 levels remained unchanged in all three fractions of HeLa cells (E–G). Furthermore, the knockdown of GRN increased TSG101 levels in the P1 and P2 EV fractions but decreased them in the cell fraction (E and H). In addition, the knockdown of GRN increased LC3-II levels in the cell and EV fractions (E and I). Importantly, in NSC-34 cells, which exhibit a lower abundance of autophagosome-like particles (F), the knockdown of Grn increased cleaved TDP-43 levels in the P2 EV fraction, mimicking the effects observed in HeLa cells (E and F). To examine the role of autophagy in the GRN knockdown–induced secretion of TDP-43, we conducted assays using ATG16L1 KO HeLa cells (J). Without the expression of ATG16L1, the knockdown of GRN no longer induced the secretion of the full-length and cleaved TDP-43 proteins into the P1 and P2 EV fractions. On the other hand, it significantly increased TSG101 levels in the EV fraction, even in ATG16L1-deficient cells (J–M).PGRN regulates the autophagy-lysosomal pathway, and its insufficiency causes FTLD-TDP , 40, 55.sed DALG , A and Br ORANGE , C and DLa cells , E–G. Fufraction , E and Hractions , E and Iarticles F, the knLa cells . TherefoLa cells , E and FLa cells J. Withount cells , J–M.FigA). In WT MEFs, PGRN localized at vesicle-like structures  and was detected in the cell and EV fractions . Notably, the molecular weight of PGRN was higher in the P1 and P2 EV fractions than in the cell fraction . The deglycosylation of the P1 and P2 EV fractions using PNGase F resulted in a downward shift of the PGRN band to a similar position to that of the cell fraction . Therefore, PGRN included in EV was highly glycosylated. The loss of PGRN expression significantly reduced full-length TDP-43 levels in the cell fraction and increased cleaved TDP-43 levels in the P2 EV fraction . The level of LC3-II, but not TSG101, was elevated in all three fractions of PGRN-null cells . These results suggest that the PGRN insufficiency increased the secretion of TDP-43 in an autophagy-dependent manner.To further validate the role of PGRN in the secretion of cleaved TDP-43 and LC3-II, we used MEFs derived from PGRN WT or KO mice A. In WT A and B) or the knockdown of TARDBP or GRN (C and D), TFEB-GFP translocated to the nucleus. In contrast, treatments that did not promote TDP-43 secretion, such as 100 μM CQ, 100 nM Vac, or the knockdown of STX17, did not lead to the nuclear localization of TFEB-GFP (A–D). Therefore, TDP-43 secretion appeared to correlate with the nuclear localization of TFEB. Conversely, we investigated whether the knockdown of TFEB interfered with the Baf-induced secretion of TDP-43. The knockdown of TFEB using siRNA decreased the levels of both full-length and cleaved TDP-43 in the P1 and P2 EV fractions, whereas it increased the levels of full-length TDP-43 in the cell fraction. Consistently, the knockdown of TFEB decreased LC3-II levels in the cell and EV fractions (E–I). These results suggest that an increase in the nuclear translocation of TFEB is a key event for the secretion of TDP-43 via EV release.The present results suggest that the inhibition of autolysosome formation followed by lysosomal acidification plays a role in the secretion of TDP-43. On the other hand, the treatment with CQ and knockdown of STX17, each of which also suppressed autolysosome formation, were insufficient to promote TDP-43 secretion, suggesting the involvement of other factors in TDP-43 secretion. We previously reported that a treatment with Baf increased the nuclear localization of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy , 56. FurP or GRN , C and DTFEB-GFP , A–D. Thractions , E–I. ThIn the brains of ALS and FTLD-TDP patients, the pathological accumulation of TDP-43 is initially deposited in specific areas and then spreads widely throughout the brain with disease progression . The C-tvia EV was regulated by autophagy. Inhibitors of v-ATPase, such as Baf and Con, suppressed autophagy but facilitated the secretion of endogenous TDP-43. On the other hand, MG, an inhibitor of proteasome degradation, facilitated autophagy and did not affect the secretion of endogenous TDP-43. In contrast, Iguchi et al. , polyclonal rabbit anti-C-terminal TDP-43 antibody , polyclonal rabbit anti-N-terminal TDP-43 antibody , polyclonal rabbit anti-PGRN antibody , polyclonal rabbit anti-TSG101 antibody , polyclonal rabbit anti-ACTN4 antibody , polyclonal rabbit anti-ADAM10 antibody , and polyclonal rabbit anti-TFEB antibody  were purchased from ProteinTech; a monoclonal mouse anti-α-tubulin antibody  and polyclonal rabbit anti-GFP antibody  from BioAcademia; a monoclonal mouse anti-LC3 antibody , normal rabbit immunoglobulin G (IgG) , polyclonal rabbit anti-Syntaxin-17 (human) antibody , and polyclonal rabbit anti-ATG16L1 antibody  from MBL; a monoclonal mouse anti-p62 antibody  from BD Biosciences; a polyclonal sheep antimouse PGRN antibody  from R&D Systems; a monoclonal mouse anti-WIPI2 antibody  from Bio-Rad; a biotin-conjugated horse antimouse IgG antibody , biotin-conjugated goat anti-rabbit IgG antibody , and biotin-conjugated rabbit antisheep IgG antibody  from Vector Laboratories; and Alexa Fluor 568-conjugated donkey antimouse IgG , and Alexa Fluor 488–conjugated donkey antisheep IgG  from Abcam.A monoclonal mouse anti-TDP-43 antibody , DALG , PlasMem Bright Green , and PlasMem Bright Red  were purchased from Dojindo; Baf , Con , and Rapa  were purchased from Adipogen; 4′,6-diamidino-2-phenylindole (DAPI) solution  and dimethyl sulfoxide (DMSO)  were purchased from Nacalai Tesque; E64d (4321-v) and pepstatin A (4397-v) were purchased from the Peptide Institute; AcidiFluor ORANGE (GC301) was purchased from Goryo Chemical; CQ diphosphate  was purchased from Fujifilm Wako; Vac  was purchased from Cayman Chemical; MG  was purchased from Enzo Life Sciences; and PNGase F  was purchased from New England Biolabs.2, and the number of passages was within 20 because a new vial was thawed. Mycoplasma contamination was tested using EZ-PCR (Sartorius).HeLa cells , HeLa cells stably expressing mRFP-GFP-LC3 , 72, ATGHeLa cells stably expressing mRFP-GFP-LC3 , 72 on c5 cells on 12-well plates cultured under the indicated conditions for 24 h were lysed in 1% (w/v) Sarkosyl in A68 -N,N,N,N-tetraacetic acid). Protein concentrations in the lysates were estimated using a BCA Protein Assay Kit (Thermo Fisher Scientific), and SDS-sample buffer was added to give a final concentration of 1% SDS. Samples were boiled for 10 min and subjected to SDS-PAGE followed by an immunoblot analysis to quantify GFP and RFP levels.HeLa cells stably expressing GFP-LC3-RFP-LC3ΔG seeded at a density of 0.5 × 102O , and 0.3% (v/v) H2O2 (FUJIFILM Wako). Images were digitized by a scanner and subjected to densitometry using ImageJ. To quantify cleaved TDP-43 bands, four cleaved TDP-43 bands surrounded by the dashed square were subjected to densitometry. The specificity of each antibody used in immunoblot analysis was validated based on the molecular weight.Immunoblotting was performed as previously reported , 67. SamImmunocytochemistry was carried out as shown previously . Cells g5 cells on coverslips (Matsunami Glass) and cultured under the indicated conditions for 24 h were fixed for 20 min with 4% paraformaldehyde in PBS and subjected to WIPI2 immunostaining to assess phagophore formation. WIPI2-positive puncta were monitored using the LSM780 confocal laser microscope equipped with a 60× objective lens. The signal intensity per cell was quantified using ImageJ and the Analyse Particles plugin . At least 33 cells in each of the three independent experiments were subjected to counting.As described in a previous study , HeLa ce5 cells on a 12-well plate cultured under the indicated conditions with or without 10 μg pepstatin A and E64d for 24 h were lysed in 1% (w/v) Sarkosyl in A68. Protein concentrations in the lysates were estimated using a BCA Protein Assay Kit, and SDS-sample buffer was added to give a final concentration of 1% SDS. Samples were boiled for 10 min and subjected to SDS-PAGE followed by immunoblotting to quantify LC3-II levels.As described in a previous study , HeLa ceHeLa cells in 12-well plates were incubated with AcidiFluor ORANGE for 2 h under the indicated conditions and subjected to live-cell imaging using the BZ-X710 fluorescence microscope (Keyence) equipped with a 20× objective lens. Five different frames of images (>335 cells) were captured, and the AcidiFluor ORANGE signal intensity per image was quantified using ImageJ with the Analyse Particles plugin . The signal intensity per cell was calculated from the total value of the signal intensity divided by the total number of cells counted from Hoechst 33342 staining. Three independent experiments were performed for quantification.et al.  Sarkosyl in A68 by sonication. Protein concentrations in cell lysates were estimated using a BCA Protein Assay Kit, and SDS-sample buffer was added to give a final concentration of 1% SDS. Regarding the fractionation of EV in culture medium, culture medium collected from the 10 cm dish was precleared by centrifugation at 800g at 4 °C for 10 min to remove nuclei and cell debris. The precleared medium was pooled in a centrifugation tube  and centrifuged at 20,000g at 4 °C for 1 h. The pellet was collected as the P1 fraction and lysed with 50 μl 2% SDS-sample buffer. Supernatants were recollected in the centrifugation tube  and centrifuged at 110,000g at 4 °C for 1 h. The pellet was collected as the P2 fraction and lysed with 50 μl of 2% SDS-sample buffer. Samples were boiled for 10 min and subjected to an immunoblot analysis.Cellular and EV fractions were prepared using the method reported by Tripisciano et al. . To prepImmunoprecipitation was performed as previously described . BrieflyImmunoelectron microscopy was conducted as previously described . The P1 m/z was employed in the positive ion mode, followed by data-dependent MS/MS using the higher-energy collisional dissociation operating mode on the top 15 ions in order of abundance. Data were analyzed with Proteome Discoverer , Mascot software , and Scaffold software . The SwissProt database (https://www.uniprot.org/) updated on July 15, 2022 and GenBank database (https://www.ncbi.nlm.nih.gov/) updated on February 27, 2020 were searched with the following parameters: monoisotopic mass, peptide mass tolerance of 10 ppm, fragment ion mass tolerance of 0.05 Da, complete tryptic digestion allowing three missed cleavages, static modification of beta-methylthiolation, variable modifications of methionine oxidation, deamidation, and pyroglutamination. A scaffold was used for label-free quantification. Peptide and protein probability thresholds of 99 and 99%, respectively, were applied. Using a target decoy approach, a peptide false discovery rate of 0.1% was selected. Data are presented in LC–MS/MS was performed according to our previously reported protocol . The P1 Proteins detected by the LC–MS/MS analysis were divided into three groups: proteins detected in the P1 fraction only, in the P2 fraction only, and in both the P1 and P2 fractions. The cellular components of proteins in these groups were examined using DAVID software .mCherry-hLC3B-pcDNA3.1  was purchased from Addgene. Full-length TDP-43 (amino acids 1–414) in EGFPC1 (Clontech), the C-terminal fragment of TDP-43 (amino acids 162–414) in EGFPC1, and the N-terminal fragment of TDP-43 (amino acids 1–273) in EGFPC1 were previously characterized , 67.To generate HeLa cells stably expressing mCherry-LC3, HeLa cells seeded on a 6 cm dish were transfected with 1 μg of a mCherry-LC3 construct and 3 μl of TransIT2020 reagent (Mirus) according to the manufacturer’s protocol. G418 -resistant clones stably expressing mCherry-LC3 were obtained by limiting dilution cloning in 96-well plates. Steady-state expression levels were confirmed by immunoblotting.TARDBP , human STX17 , human TFEB , mouse Grn , were used for the knockdown of human TARDBP, STX17, TFEB, and mouse Grn, respectively. Predesigned stealth siRNA duplexes to target human GRN  were used for the knockdown of human GRN, as previously reported (Transfection was performed as previously reported . In tranreported . The steGFP-tagged expression vectors of full-length (amino acids 1–414), the N-terminal fragment (amino acids 1–273), and C-terminal fragment (amino acids 162–414) of TDP-43 were previously prepared , 67. HeLHeLa cells were transfected with TDP-43 amino acids 162 to 414 or mCherry-LC3 for 24 h and then seeded on μ-Dish, 35 mm high (ibidi). After a 24 h stimulation with 100 nM Baf, the plasma membrane was visualized with PlasMem Bright Green or PlasMem Bright Red according to the manufacturer’s protocol. Live-cell imaging was conducted using the LSM780 confocal laser microscope equipped with a 60× objective lens.As shown in previous studies , 67, autvia Oviductal Nucleic Acids Delivery (iGONAD) , transactivating CRISPR RNA , and 1 mg/ml Cas9-NLS protein (NIPPON GENE), and electroporated using the NEPA21 electroporator (Nepagene) on embryonic day 0.7 (approximately 15:00). Newborn mice were weaned at 3 weeks old, and their genome was extracted from their tails. The alignment of the genome-edited site was identified with Sanger sequencing (Azent). Mice with the mutation p.Ala24GlyfsTer22 were selected and backcrossed with ordinary C57BL/6 mice three times. Mice were bred in the laboratory animal facility (Imabari) in accordance with the guidelines of the Institute for Laboratory Animal Research under controlled light , temperature (23 ± 1 °C), and humidity (55 ± 10%), and were given free access to food and water. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Okayama University of Science and were approved by the Institutional Animal Care and Use Committee of Okayama University of Science (permit number: 2019-047).PGRN CRISPR KO mice were generated by improved-Genome editing (iGONAD) , 78. BriThe preparation of MEFs was described in a previous study . Embryos2O. In the PNGase F-positive group, pellets were mixed with 2.5 μl GlycoBuffer 2, 20 μl H2O, and 2.5 μl PNGase F. After a 24 h incubation at 37 °C, samples were mixed with 25 μl SDS-sample buffer  and boiled for 10 min for immunoblotting.According to the manufacturer’s protocol, the N-linked glycans of proteins in the P1 and P2 fractions of WT MEFs were deglycosylated with PNGase F . Briefly, P1 and P2 pellets were prepared as shown previously. In the PNGase F-negative group (control), pellets were mixed with 2.5 μl GlycoBuffer 2  and 22.5 μl HSH-SY5Y cells stably expressing TFEB-GFP  seeded ot test, Mann–Whitney U test, or Tukey’s post hoc test were performed using Excel (Microsoft Office) or EZR  or EZR . Data arAll data are contained in the figures, figure legends, or This article contains The authors declare that they have no conflicts of interest with the contents of this article.