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Alternative pre-mRNA splicing is key to proteome diversity; however, the biological roles of alternative splicing (AS) in signaling pathways remain elusive. Here, we focus on TEA domain transcription factor 1 (TEAD1), a YAP binding factor in the Hippo signaling pathway. Public database analyses showed that expression of YAP-TEAD target genes negatively correlated with the expression of a TEAD1 isoform lacking exon 6 (TEAD1ΔE6) but did not correlate with overall TEAD1 expression. We confirmed that the transcriptional activity and oncogenic properties of the full-length TEAD1 isoform were greater than those of TEAD1ΔE6, with the difference in transcription related to YAP interaction. Furthermore, we showed that RNA-binding Fox-1 homolog 2 (RBFOX2) promoted the inclusion of TEAD1 exon 6 via binding to the conserved GCAUG element in the downstream intron. These results suggest a regulatory mechanism of RBFOX2-mediated TEAD1 AS and provide insight into AS-specific modulation of signaling pathways.

Human lung organoids (hLOs) are useful for disease modelling and drug screening. However, a lack of immune cells in hLOs limits the recapitulation of in vivo cellular physiology. Here, we generated hLOs containing alveolar macrophage (AMφ)-like cells derived from pluripotent stem cells (PSC). To bridge hLOs with advanced human lung high-resolution X-ray computed tomography (CT), we acquired quantitative micro-CT images. Three hLO types were observed during differentiation. Among them, alveolar hLOs highly expressed not only lung epithelial cell markers but also AMφ-specific markers. Furthermore, CD68

Bee venom is a medicinal product that is widely used in traditional therapies owing to its excellent anti-inflammatory activity. However, the use of bee venom has shown adverse effects. Therefore, there is a need for research that can remove the cytotoxicity of bee venom and enhance its efficacy. In this study, we hydrolyzed melittin, the main component of bee venom, and removed the other components to eliminate the toxicity of bee venom. To compare the efficacy of bee venom and detoxified bee venom, we examined their antioxidant effects using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. In addition, cytotoxicity was confirmed in MCF 10A and RAW 264.7 cells, using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. Detoxified bee venom showed a strong antioxidant activity and decreased a cytotoxicity in MCF 10A and RAW 264.7 cells. The anti-inflammatory activity of detoxified bee venom and bee venom were assessed by comparison of the expression of inflammatory cytokine mRNA and phosphorylation of IκBα in RAW 264.7 cells. Degranulation in RBL-2H3 cells was analyzed through β-hexosaminidase release assay to confirm the allergenic activity of bee venom and detoxified bee venom. Treatment of the detoxified bee venom inhibited inflammatory cytokine mRNA expression, IκBα phosphorylation, and β-hexosaminidase release. Taken together, the results indicated that compared to bee venom, detoxified bee venom exhibited decreased cytotoxicity and allergenicity and increased anti-inflammatory activity. In conclusion, detoxification of bee venom efficiently decreases the adverse effects, making it suitable for medicinal applications. Supplementary Information The online version contains supplementary material available at 10.1007/s12010-021-03653-2.

The autophagy-mediated lysosomal pathway plays an important role in conferring stress tolerance to tumor cells during cellular stress such as increased metabolic demands. Thus, targeted disruption of this function and inducing lysosomal cell death have been proved to be a useful cancer therapeutic approach. In this study, we reported that octyl syringate (OS), a novel phenolic derivate, was preferentially cytotoxic to various cancer cells but was significantly less cytotoxic to non-transformed cells. Treatment with OS resulted in non-apoptotic cell death in a caspase-independent manner. Notably, OS not only enhanced accumulation of autophagic substrates, including lapidated LC3 and sequestosome-1, but also inhibited their degradation via an autophagic flux. In addition, OS destabilized the lysosomal function, followed by the intracellular accumulation of the non-digestive autophagic substrates such as bovine serum albumin and stress granules. Furthermore, OS triggered the release of lysosomal enzymes into the cytoplasm that contributed to OS-induced non-apoptotic cell death. Finally, we demonstrated that OS was well tolerated and reduced tumor growth in mouse xenograft models. Taken together, our study identifies OS as a novel anticancer agent that induces lysosomal destabilization and subsequently inhibits autophagic flux and further supports development of OS as a lysosome-targeting compound in cancer therapy. • Octyl syringate, a phenolic derivate, is preferentially cytotoxic to various cancer cells. • Octyl syringate destabilizes the lysosomal function. • Octyl syringate blocks the autophagic flux. • Octyl syringate is a potential candidate compound for cancer therapy.

Cheese contains various beneficial nutrients, including calcium and whey protein, as well as large amounts of saturated fatty acids. Thus, intake of cheese increases the production of low‐density lipoprotein‐cholesterol (LDL‐C), a well‐defined risk factor for cardiovascular disease. Therefore, identification of natural products that inhibit LDL‐C production following cheese intake and verification of the efficacy of such products in animal models are essential. Here, we evaluated the effects of Allium hookeri, a well‐known traditional herbal remedy, on metabolism and thermogenesis in mice consuming a cheese‐containing diet. Intake of A. hookeri extracts significantly blocked increases in body weight and fat mass caused by intake of Gouda cheese in mice. Additionally, increases in blood triglyceride levels following intake of Gouda cheese were alleviated by A. hookeri. Moreover, intake of Gouda cheese enhanced thermogenesis efficiency. Thus, A. hookeri may have applications as an important additive for reducing the risk of metabolic disease resulting from cheese consumption.

Brucite, a mineral form of magnesium hydroxide, has been used industrially as a useful refractory insulator. However, its antibacterial activity remains largely unknown. This study investigated the antibacterial activity of brucite in two bacterial strains, Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive). Brucite, compared to other minerals, showed a strong antibacterial activity against both bacterial strains. The surface structure of brucite examined using scanning electron microscopy (SEM) had no implications in its antibacterial activity. Brucite in lysogeny broth (LB) medium maintained the basicity (pH 9) of the solution for a prolonged period of time. The basicity of the medium was responsible for the antibacterial activity of brucite. The study results identified brucite as a potent antibacterial agent that required no further processing and also revealed the underlying mechanism of action responsible for its antibacterial activity.

Magnesium is well known as a biodegradable biomaterial that has been reported to promote bone remodeling in several studies; however, the underlying biological mechanism remains unclear. In the present study, the role of magnesium ions in the migration of U-2 OS cells, which are osteoblast-like cell lines, was investigated. Magnesium treatment did not significantly alter the global transcriptome of U-2 OS cells, but increased the protein expression level of SNAI2, an epithelial-mesenchymal transition (EMT) marker. In addition, it was confirmed that the junctional site localization of Zona-occludens 1 (ZO-1), a representative tight junction protein, was destroyed by magnesium treatment; furthermore, it was determined that cytoplasmic localization increased, and alkaline phosphatase (ALP) activity increased. The obtained results on the mechanism by which magnesium is involved in osteoblast migration, which is important for fracture healing, will contribute to the understanding of the bone-formation process in patients with osteoporosis and musculoskeletal injury.

Calcific tendinopathy (CT), developed due to calcium hydroxyapatite deposition in the rotator cuff tendon, mostly affects women in their 40 s and 50 s and causes severe shoulder pain. However, the molecular basis of its pathogenesis and appropriate treatment methods are largely unknown. In this study, we identified 202 differentially expressed genes (DEGs) between calcific and adjacent normal tendon tissues of rotator cuff using RNA sequencing-based transcriptome analysis. The DEGs were highly enriched in extracellular matrix (ECM) degradation and inflammation-related processes. Further, matrix metalloproteinase 9 (MMP9) and matrix metalloproteinase 13 (MMP13), two of the enzymes associated with ECM degradation, were found to be highly upregulated 25.85- and 19.40-fold, respectively, in the calcific tendon tissues compared to the adjacent normal tendon tissues. Histopathological analyses indicated collagen degradation and macrophage infiltration at the sites of calcific deposit in the rotator cuff tendon. Our study acts as a foundation that may help in better understanding of the pathogenesis associated with CT, and thus in better management of the disease.

Exposure to atmospheric particulate matter (PM) increases morbidity and mortality in respiratory diseases by causing various adverse health effects; however, the effects of PM exposure on cellular stress under virus-infected conditions remain unclear. The effects of PM under 10 μm (PM

The underlying mechanisms of splicing regulation through non-canonical splice junction processing remain largely unknown. Here, we identified two RBFOX2 splicing isoforms by alternative 3' splice site selection of exon 9; the non-canonical splice junction processed RBFOX2 transcript (RBFOX2-N.C.) was expressed by the selection of the 3' splice GG acceptor sequence. The cytoplasmic localization of RBFOX2-C., a canonical splice junction-processed RBFOX2 transcript, was different from that of RBFOX2-N.C., which showed nuclear localization. In addition, we confirmed that RBFOX2-C. showed a significantly stronger localization into stress granules than RBFOX2-N.C. upon sodium arsenite treatment. Next, we investigated the importance of non-canonical 3' splice GG sequence selection of specific cis-regulatory elements using minigene constructs of the RBFOX2 gene. We found that the non-canonical 3' splice GG sequence and suboptimal branch point site adjacent region were critical for RBFOX2-N.C. expression through a non-canonical 3' splice selection. Our results suggest a regulatory mechanism for the non-canonical 3' splice selection in the RBFOX2 gene, providing a basis for studies related to the regulation of alternative pre-mRNA splicing through non-canonical splice junction processing.

Bisphenol-A (BPA) is a representative endocrine disruptor, widely used in a variety of products including plastics, medical equipment and receipts. Hence, most people are exposed to BPA via the skin, digestive system or inhalation in everyday life. Furthermore, BPA crosses the blood–brain barrier and is linked to multiple neurological dysfunctions found in neurodegenerative and neuropsychological disorders. However, the mechanisms underlying BPA-associated neurological dysfunctions remain poorly understood. Here, we report that BPA exposure alters synapse morphology and function in the cerebral cortex. Cortical pyramidal neurons treated with BPA showed reduced size and number of dendrites and spines. The density of excitatory synapses was also decreased by BPA treatment. More importantly, we found that BPA disrupted normal synaptic transmission and cognitive behavior. RGS4 and its downstream BDNF/NTRK2 pathway appeared to mediate the effect of BPA on synaptic and neurological function. Our findings provide molecular mechanistic insights into anatomical and physiological neurotoxic consequences related to a potent endocrine modifier.

Studies on the discovery and function of antioxidants are consistently being performed because oxidative stress can cause various diseases. Many compounds and natural products have antioxidant activity in vitro; however, it is often difficult to reproduce their effects in vivo. Additionally, methods to measure antioxidant activities in cells are also scarce. Here, we investigated the antioxidant activity of milk proteins by observing the formation of arsenite-induced stress granules as a tool to evaluate antioxidant activity in cells. Milk proteins not only decreased the formation of stress granules in several cell types but also scavenged 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cations in vitro. In addition, milk proteins inhibited cellular senescence based on an SA-β-galactosidase assay, and increased differentiation to myotubes from myoblasts isolated from the skeletal muscles of mouse pups. Taken together, our results demonstrate that milk proteins have an antiaging effect, especially prevention of skeletal muscle loss, through their antioxidant activities. PRACTICAL APPLICATION: Our results provide that antioxidant effects of milk proteins containing α-caseins, β-caseins, and β-lactoglobulin can mitigate aging-related damage induced by oxidative stress through showing inhibition of cellular senescence and increase of differentiation and maturation of myoblast. Therefore, we suggest that milk proteins could be potent health supplements to prevent aging-associated diseases, especially sarcopenia.

Although the incidence of severe fever with thrombocytopenia syndrome virus (SFTSV) infection has increased from its discovery with a mortality rate of 10–20%, no effective vaccines are currently available. Here we describe the development of a SFTSV DNA vaccine, its immunogenicity, and its protective efficacy. Vaccine candidates induce both a neutralizing antibody response and multifunctional SFTSV-specific T cell response in mice and ferrets. When the vaccine efficacy is investigated in aged-ferrets that recapitulate fatal clinical symptoms, vaccinated ferrets are completely protected from lethal SFTSV challenge without developing any clinical signs. A serum transfer study reveals that anti-envelope antibodies play an important role in protective immunity. Our results suggest that Gn/Gc may be the most effective antigens for inducing protective immunity and non-envelope-specific T cell responses also can contribute to protection against SFTSV infection. This study provides important insights into the development of an effective vaccine, as well as corresponding immune parameters, to control SFTSV infection., Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging tick-borne virus with no specific treatment or vaccine available. Here, the authors develop a DNA vaccine for SFTSV that is protective against lethal challenge in ferrets and show that anti-envelope antibodies are important for protection.

Stress granules (SGs) are stalled translation initiation complexes comprising untranslated mRNAs and RNA-binding proteins (RBPs). RBP fox-1 homolog 2 (Rbfox2), a component of SGs, binds to retinoblastoma 1 (RB1) mRNA, which is closely related to cancer progression; however, the role of Rbfox2 in cancer progression remains largely unknown. In this study, we confirmed that Rbfox2, which is present in the nucleus as a splicing regulator, localizes to the cytoplasm of human colon cancer tissues and that induction of Rbfox2 dissociation from SGs by resveratrol treatment inhibits cancer progression. We also observed that Rbfox2 in SGs inhibited RB1 protein expression and promoted cell cycle progression. Additionally, resveratrol treatment inhibited SG-mediated Rbfox2 localization, further inhibiting RB1 protein expression, and inhibited specific Rbfox2 localization to the cytoplasm in melanoma B16-F10 cells, thereby effectively inhibiting metastasis and tumor growth ability. These results indicate that Rbfox2 dissociation from SGs attenuates cancer progression and offer insight into the mechanism associated with Rbfox2 dissociation, thereby marking Rbfox2 as a potential candidate target for cancer therapy., Cancer: Interfering with RNA processing to prevent tumor growth Resveratrol, an antioxidant found in red grapes, slows cancer progression by interfering with the localization and function of the RNA-binding protein Rbfox2. A study led by Kee Kim at Chungnam National University and Su-Hyung Park at Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea, showed that in human colon cancer cells Rbfox2 is located in the cytoplasm where it promotes cell proliferation by blocking the assembly of the tumor suppressor protein RB1. Treatment with resveratrol prevented the migration of Rbfox2 from the nucleus to the cytoplasm and significantly reduced tumor growth in a mouse model of melanoma. This study not only sheds light on the protective effects of resveratrol but also suggests that Rbfox2 could be a potential target for the development of new anticancer drugs.

In the 3D-printed polycaprolactone/hydroxyapatite composite scaffold, hydroxyapatite particles are usually covered by a thin-film of polycaprolactone because of the rheological characteristics of the extrusion process. This phenomenon could disrupt the original bioactive characteristics of hydroxyapatite particles. In this study, to expose the hydroxyapatite particles covered by a thin-film of polycaprolactone, an alkaline erosion method was proposed. Moreover, to investigate the cell activity and biomimetic mineralization influenced by hydroxyapatite exposure, polycaprolactone scaffolds, polycaprolactone scaffolds with alkaline erosion, and polycaprolactone/hydroxyapatite scaffolds were fabricated as control groups and compared with the polycaprolactone/hydroxyapatite scaffolds with alkaline erosion. Furthermore, to characterize the 3D-printed composite scaffold in terms of hydroxyapatite exposure, several assessments were made including morphology, pore size, porosity, mechanical compressive modulus, surface roughness, water absorption. Consequently, the proposed alkaline erosion for hydroxyapatite particle exposure had little effect on the structure of the fabricated scaffolds via the 3D-printing system including the designed pore size, porosity, and mechanical properties. Moreover, mechanical properties of the polycaprolactone/hydroxyapatite scaffolds were increased by the high dispersion of hydroxyapatite in the polycaprolactone matrix. Additionally, we verified that the exposure of hydroxyapatite particles by alkaline erosion improves cell proliferation and biomimetic mineralization, because calcium and phosphate ions were rapidly deposited on the scaffold.

Sarcopenia is a disease characterized by the loss of muscle mass and function that occurs mainly in older adults. The present study was designed to investigate the hypothesis that water extract of Lycium chinense (WELC) would improve muscle function and promote myogenesis for sarcopenia. We investigated the effect of water extracts of L. chinense on muscular endurance function and myogenesis to examine its efficacy in sarcopenia. Intake of WELC‐containing cheese enhanced the muscular endurance function of mice in treadmill endurance tests. In addition, the cross‐sectional areas of muscle fibers in the gastrocnemius muscle of L. chinense‐fed mice were greater than that of control mice. Furthermore, WELC and its key component marker substance betaine promoted myogenesis of myoblasts by increasing the expression of the myogenic protein myosin heavy chain 3 (Myh3) and myotube formation. Taken together, our results suggest that L. chinense may potentially be useful in the development of preventive and therapeutic agents for sarcopenia, as well as in providing basic knowledge on myogenesis and muscular functions., WELC and its key component marker substance betaine promoted myogenesis of myoblasts by increasing the expression of the myogenic protein myosin heavy chain 3 (Myh3) and myotube formation. L. chinense may potentially be useful in the development of preventive and therapeutic agents for sarcopenia, as well as in providing basic knowledge on myogenesis and muscular functions.

Early-life exposure to bisphenol A (BPA), synthetic compound used in polycarbonate plastic, is associated with altered cognitive and emotional behavior later in life. However, the brain mechanism underlying the behavioral deficits is unknown. Here, we show that maternal BPA exposure disrupted self-renewal and differentiation of neural progenitors during cortical development. The BPA exposure reduced the neuron number, whereas it increased glial cells in the cerebral cortex. Also, synaptic formation and transmission in the cerebral cortex were suppressed after maternal BPA exposure. These changes appeared to be associated with autophagy as a gene ontology analysis of RNA-seq identified an autophagy domain in the BPA condition. Mouse behavioral tests revealed that maternal BPA caused hyperactivity and social deficits in adult offspring. Together, these results suggest that maternal BPA exposure leads to abnormal cortical architecture and function likely by activating autophagy.

Cheese contains various beneficial nutrients, including calcium and whey protein, as well as large amounts of saturated fatty acids. Thus, intake of cheese increases the production of low‐density lipoprotein‐cholesterol (LDL‐C), a well‐defined risk factor for cardiovascular disease. Therefore, identification of natural products that inhibit LDL‐C production following cheese intake and verification of the efficacy of such products in animal models are essential. Here, we evaluated the effects of Allium hookeri, a well‐known traditional herbal remedy, on metabolism and thermogenesis in mice consuming a cheese‐containing diet. Intake of A. hookeri extracts significantly blocked increases in body weight and fat mass caused by intake of Gouda cheese in mice. Additionally, increases in blood triglyceride levels following intake of Gouda cheese were alleviated by A. hookeri. Moreover, intake of Gouda cheese enhanced thermogenesis efficiency. Thus, A. hookeri may have applications as an important additive for reducing the risk of metabolic disease resulting from cheese consumption., Identification of natural products that inhibit LDL‐C production following cheese intake and verification of the efficacy of such products in animal models are essential. Intake of Allium hookeri extracts significantly blocked increases in body weight and fat mass caused by intake of Gouda cheese in mice. Moreover, intake of Gouda cheese enhanced thermogenesis efficiency. Thus, A. hookeri may have applications as an important additive for reducing the risk of metabolic disease resulting from cheese consumption.

Brucite, a mineral form of magnesium hydroxide, has been used industrially as a useful refractory insulator. However, its antibacterial activity remains largely unknown. This study investigated the antibacterial activity of brucite in two bacterial strains

Osteochondral defects, including damage to both the articular cartilage and the subchondral bone, are challenging to repair. Although many technological advancements have been made in recent years, there are technical difficulties in the engineering of cartilage and bone layers, simultaneously. Moreover, there is a great need for a valuable in vitro platform enabling the assessment of osteochondral tissues to reduce pre-operative risk. Three-dimensional (3D) bioprinting systems may be a promising approach for fabricating human tissues and organs. Here, we aimed to develop a polycaprolactone (PCL)/alginate bipartite hybrid scaffold using a multihead 3D bioprinting system. The hybrid scaffold was composed of PCL, which could improve the mechanical properties of the construct, and alginate, encapsulating progenitor cells that could differentiate into cartilage and bone. To differentiate the bipartite hybrid scaffold into osteochondral tissue, a polydimethylsiloxane coculture system for osteochondral tissue (PCSOT) was designed and developed. Based on evaluation of the biological performance of the novel hybrid scaffold, the PCL/alginate bipartite scaffold was successfully fabricated, importantly, our findings suggest that this PCSOT system may be applicable as an in vitro platform for osteochondral tissue engineering.

Protein-protein interactions are essential in various cellular processes including regulation of gene expression, formation of protein complexes, and cellular signaling transduction. In particular, several proteins in the nucleus interact to regulate transcription and RNA splicing. These protein-protein interactions are short and weak and occur through transient processes, making it difficult to identify these interactions. In addition, detection of interacting partners in vitro using cell lysates cannot provide complete information due to the loss of spatial organization and changes in protein modification. Here we describe an in vivo crosslinking technique using disuccinimidyl suberate (DSS), which is useful to capture and stabilize proteins to analyze the interacting proteins.

Dysfunction of tight junctions and their components can cause diverse skin diseases. Here, we investigated the expression of claudin 1, a major tight junction protein, and changes of tight junction capacity upon treatment of the extracts of Cudrania tricuspidata (C. tricuspidata) and its components, chlorogenic acid, kaempferol, and quercetin. The effects of ethanol extracts of C. tricuspidata (EECT) and water extracts of C. tricuspidata (WECT) on the viability of human keratinocyte HaCaT cells were assessed by cell proliferation assay. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was conducted to measure the expression of claudin 1 mRNA. The protein expression of claudin 1 was analyzed by western blot and its tight junctional distribution was observed with immunofluorescence microscopy analysis. The tight junction capacity was analyzed by dispase assay. Upon treatment of WECT to HaCaT cells, the mRNA and protein expressions of claudin 1 were increased. In addition, chlorogenic acid, kaempferol, and quercetin increased claudin 1 protein expression levels in a dose-dependent manner. WECT and these three compounds enhanced the tight junction capacity of HaCaT cells in dispase assay. WECT, and its components, such as chlorogenic acid, kaempferol, and quercetin, upregulates both mRNA and protein expressions of claudin 1, which leads to the enhancement of tight junction capacity. Thus, WECT could be a therapeutic approach for treating tight junction-disrupted conditions such as atopic dermatitis and psoriasis.

Protein-protein interactions are essential in various cellular processes including regulation of gene expression, formation of protein complexes, and cellular signaling transduction. In particular, several proteins in the nucleus interact to regulate transcription and RNA splicing. These protein-protein interactions are short and weak and occur through transient processes, making it difficult to identify these interactions. In addition, detection of interacting partners in vitro using cell lysates cannot provide complete information due to the loss of spatial organization and changes in protein modification. Here we describe an in vivo crosslinking technique using disuccinimidyl suberate (DSS), which is useful to capture and stabilize proteins to analyze the interacting proteins.

Alternative splicing is an essential process in eukaryotes, as it increases the complexity of gene expression by generating multiple proteins from a single pre-mRNA. However, information on the regulatory mechanisms for alternative splicing is lacking, because splicing occurs over a short period via the transient interactions of proteins within functional complexes of the spliceosome. Here, we investigated in detail the molecular mechanisms connecting alternative splicing with epigenetic mechanisms. We identified interactions between histone proteins and splicing factors such as Rbfox2, Rbfox3, and splicing factor proline and glutamine rich protein (SFPQ) by in vivo crosslinking and immunoprecipitation. Furthermore, we confirmed that splicing factors were bound to specific modified residues of histone proteins. Additionally, changes in histone methylation due to histone methyltransferase inhibitor treatment notably affected alternative splicing in selected genes. Therefore, we suggested that there may be crosstalk mechanisms connecting histone modifications and RNA-binding proteins that increase the local concentration of RNA-binding proteins in alternative exon loci of nucleosomes by binding specific modified histone proteins, leading to alternative splicing. This crosstalk mechanism may play a major role in epigenetic processes such as histone modification and the regulation of alternative splicing.

Rbfox family of proteins that consists of Rbfox1, Rbfox2, and Rbfox3 in mammals regulates alternative pre-mRNA splicing in various tissues via direct binding to their RNA binding element. Although many studies have indicated the splicing activity of each member of the Rbfox family, the interactions of Rbfox family proteins are largely unknown. Here, we have investigated interactions among Rbfox family proteins. Co-immunoprecipitation (Co-IP) and GST-pull down assays confirmed that Rbfox proteins form homo and hetero complexes. Moreover, in vivo crosslinking using disuccinimidyl suberate treatment indicated that the Rbfox proteins form a dimer which then assembles with other proteins to form a large multiprotein complex. Duolink in situ proximity ligation (PLA) assay revealed that neuron specific Rbfox3 protein interacts with other Rbfox family proteins. This study is the first to provide an evidence that Rbfox family proteins form homo- and hetero-oligomeric complexes in vivo.

Claudin 1, a major tight junction protein, is highly expressed in various types of tumors such as thyroid, breast, and colorectal cancers. Moreover, claudin 1 is frequently found in the cytoplasm in various types of tumor cells. However, the cytoplasmic function of claudin 1 in tumors still remains largely unknown. Here, we investigated the novel function of cytoplasmic claudin 1 in autophagy. The mRNA expression level of claudin 1 was higher in several types of tumors than in normal tissues. Furthermore, colon tumor tissues showed increased autophagy compared with the adjacent normal tissues. Both endogenous and exogenous claudin 1 showed a cytoplasmic punctate staining pattern and were co-stained with the lysosome-associated membrane protein 1 (LAMP1). Importantly, autophagy-induced conditions, including starvation, increased the protein stability of claudin 1. Moreover, the increased level of claudin 1 stimulated autophagy by decreasing the level of the autophagy substrate, sequestosome1/p62 (SQSTM1), under autophagy-inducing conditions; activation of AMP-activated protein kinase (AMPK) and inhibition of mammalian target of rapamycin (mTOR). Taken together, we demonstrate that the novel function of cytoplasmic claudin 1 is related to autophagy. This study is the first to show a cytoplasmic function of claudin 1 as an autophagy regulator and provides the evidence that claudin 1-mediated autophagy regulation is an integral part of the mechanism by which claudin 1 regulates cancer progression.