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In this study, we conducted large-scale experiments with novel descriptions of determinism. Our goal was to investigate the effects of desires for punishment and comprehension errors on people's intuitions about free will and moral responsibility in deterministic scenarios. Previous research has acknowledged the influence of these factors, but their total effect has not been revealed. Using a large-scale survey of Japanese participants, we found that the failure to understand causal determination (intrusion) has limited effects relative to other factors and that the conflation of determinism and epiphenomenalism (bypassing) has a significant influence, even when controlling for other variables. This leads to the increased prevalence of incompatibilist responses. Furthermore, our results demonstrated a close association between the attribution of free will/responsibility and retributive desire. While further research is needed to establish the causal relationship between these factors, this association is consistent with Cory Clark and colleagues' study that increased desire contributes to increased compatibilist responses and their claim that a definitive intuition about free will may be elusive., (© 2024 Cognitive Science Society LLC.)

While microplastics (MPs) have emerged as a significant threat, information on MPs <330 μm (SMPs) is limited by the lack of simple quantification methods. We examined the potential application of salps, non-selective filter-feeding tunicates, to estimate in-situ SMP densities. After collection, salp guts were dissected, dissolved, and filtered to analyze MPs using μFTIR. The results showed each gut samples contained 1.96 ± 1.49 MP particles; their polymer composition and size were consistent with those in ambient seawater. When the SMP quantity in salp gut was converted to in-situ densities using previously published feeding parameters, SMP densities ranged between 235 and 1209 particles/m 3 ; they were strongly correlated with those in seawater. Importantly, this method, which is less labor intensive than other methods, could easily characterize in-situ SMP distribution of different marine environments, thus improve the monitoring of their pollution. Furthermore, it could be applied to examine historical contributions of SMPs using archived salp samples., Competing Interests: Declaration of competing interest The authors declare no competing interests regarding the work described in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest for this article.

Transforming growth factor β (TGF-β) is a pleiotropic cytokine that is widely distributed throughout the body. Its receptor proteins, TGF-β type I and type II receptors, are also ubiquitously expressed. Therefore, the regulation of various signaling outputs in a context-dependent manner is a critical issue in this field. Smad proteins were originally identified as signal-activated transcription factors similar to signal transducer and activator of transcription proteins. Smads are activated by serine phosphorylation mediated by intrinsic receptor dual specificity kinases of the TGF-β family, indicating that Smads are receptor-restricted effector molecules downstream of ligands of the TGF-β family. Smad proteins have other functions in addition to transcriptional regulation, including post-transcriptional regulation of micro-RNA processing, pre-mRNA splicing, and m 6 A methylation. Recent technical advances have identified a novel landscape of Smad-dependent signal transduction, including regulation of mitochondrial function without involving regulation of gene expression. Therefore, Smad proteins are receptor-activated transcription factors and also act as intracellular signaling modulators with multiple modes of function. In this review, we discuss the role of Smad proteins as receptor-activated transcription factors and beyond. We also describe the functional differences between Smad2 and Smad3, two receptor-activated Smad proteins downstream of TGF-β, activin, myostatin, growth and differentiation factor (GDF) 11, and Nodal., Competing Interests: Conflicts of interest The authors declare no conflicts of interest with the content of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This article critically examines the experimental philosophy of free will, particularly the interplay between ordinary individuals' compatibilist and incompatibilist intuitions. It explores key insights from research studies that propose "natural compatibilism" and "natural incompatibilism". These studies reveal a complex landscape of folk intuitions, where participants appear to exhibit both types of intuitions. Here, we examine error theories, which purport to explain the coexistence of apparently contradictory intuitions: the Affective Performance Error hypothesis, the "Free Will No Matter What" hypothesis, the Bypassing hypothesis, and the Intrusion hypothesis, and the article explores the cognitive errors that could shape individuals' inconsistent perceptions of free will. We then explore three possibilities regarding folk intuitions: most individuals may hold either compatibilist or incompatibilist intuitions, both simultaneously, or neither. Our aim is to deepen the understanding of the complex dynamics of intuitions about free will, and we close with suggestions for future studies in experimental philosophy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Inarimori, Honma and Miyazono.)

Since various imaging modalities have been developed, cancer metastasis can be detected from an early stage. However, limitations still exist, especially in terms of spatial resolution. Tissue-clearing technology has emerged as a new imaging modality in cancer research, which has been developed and utilized for a long time mainly in neuroscience field. This method enables us to detect cancer metastatic foci with single-cell resolution at whole mouse body/organ level. On top of that, 3D images of cancer metastasis of whole mouse organs make it easy to understand their characteristics. Recently, further applications of tissue clearing methods were reported in combination with reporter systems, labeling, and machine learning. In this review, we would like to provide an overview of this technique and current applications in cancer research and discuss their potentials and limitations., Competing Interests: The authors have no conflict of interest to disclose., (© 2024 The Author(s). Published by Upsala Medical Society.)

The tumor microenvironment (TME) consists of cancer cells surrounded by stromal components including tumor vessels. Transforming growth factor-β (TGF-β) promotes tumor progression by inducing epithelial-mesenchymal transition (EMT) in cancer cells and stimulating tumor angiogenesis in the tumor stroma. We previously developed an Fc chimeric TGF-β receptor containing both TGF-β type I (TβRI) and type II (TβRII) receptors (TβRI-TβRII-Fc), which trapped all TGF-β isoforms and suppressed tumor growth. However, the precise mechanisms underlying this action have not yet been elucidated. In the present study, we showed that the recombinant TβRI-TβRII-Fc protein effectively suppressed in vitro EMT of oral cancer cells and in vivo tumor growth in a human oral cancer cell xenograft mouse model. Tumor cell proliferation and angiogenesis were suppressed in tumors treated with TβRI-TβRII-Fc. Molecular profiling of human cancer cells and mouse stroma revealed that K-Ras signaling and angiogenesis were suppressed. Administration of TβRI-TβRII-Fc protein decreased the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), interleukin-1β (IL-1β) and epiregulin (EREG) in the TME of oral cancer tumor xenografts. HB-EGF increased proliferation of human oral cancer cells and mouse endothelial cells by activating ERK1/2 phosphorylation. HB-EGF also promoted oral cancer cell-derived tumor formation by enhancing cancer cell proliferation and tumor angiogenesis. In addition, increased expressions of IL-1β and EREG in oral cancer cells significantly enhanced tumor formation. These results suggest that TGF-β signaling in the TME controls cancer cell proliferation and angiogenesis by activating HB-EGF/IL-1β/EREG pathways and that TβRI-TβRII-Fc protein is a promising tool for targeting the TME networks., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

The distribution of floating plastic debris around the Kuroshio Current which transports plastics from the coastal waters of Asian countries to North Pacific subtropical gyre, was investigated in 2014. The mean abundance and weight of plastic debris on the sea surface were 100,376 counts/km 2 and 446.16 g/km 2 , respectively. Intensive plastic accumulation was observed in the frontal area between the northern edge of the Kuroshio and coastal waters off Shikoku, while a relatively higher abundance in the south of Kuroshio was generally associated with anticyclonic mesoscale eddies. Such an accumulation resulted from the eddy-Kuroshio interactions which are specifically associated with the offshore non-large meandering Kuroshio path. Overall, white, fragmented, small-sized (≤1 mm) particles with polyethylene and polypropylene polymers were dominant. In the southern area of Kuroshio, the contribution of polystyrene and larger-sized plastic was higher, suggesting a rapid influx of fresh particles from western Japan to offshore by the northwest monsoon., Competing Interests: Declaration of competing interest The authors declare no competing interests regarding the work described in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The red blood cells (RBCs) of vertebrates have evolved into two basic shapes, with nucleated nonmammalian RBCs having a biconvex ellipsoidal shape and anuclear mammalian RBCs having a biconcave disk shape. In contrast, camelid RBCs are flat ellipsoids with reduced membrane deformability, suggesting altered membrane skeletal organization. However, the mechanisms responsible for their elliptocytic shape and reduced deformability have not been determined. We here showed that in alpaca RBCs, protein 4.1R, a major component of the membrane skeleton, contains an alternatively spliced exon 14-derived cassette (e14) not observed in the highly conserved 80 kDa 4.1R of other highly deformable biconcave mammalian RBCs. The inclusion of this exon, along with the preceding unordered proline- and glutamic acid-rich peptide (PE), results in a larger and unique 90 kDa camelid 4.1R. Human 4.1R containing e14 and PE, but not PE alone, showed markedly increased ability to form a spectrin-actin-4.1R ternary complex in viscosity assays. A similar facilitated ternary complex was formed by human 4.1R possessing a duplication of the spectrin-actin-binding domain, one of the mutations known to cause human hereditary elliptocytosis. The e14- and PE-containing mutant also exhibited an increased binding affinity to β-spectrin compared with WT 4.1R. Taken together, these findings indicate that 4.1R protein with the e14 cassette results in the formation and maintenance of a hyperstable membrane skeleton, resulting in rigid red ellipsoidal cells in camelid species, and suggest that membrane structure is evolutionarily regulated by alternative splicing of exons in the 4.1R gene., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

This study aimed to evaluate the effects of jaw-opening exercises with and without pain on temporomandibular disorders (TMDs), specifically in relation to pain intensity and range-of-mouth opening in patients with TMDs. Participants were randomly assigned to either the jaw-opening exercise with pain (JE w/pain) or the jaw-opening exercise without pain (JE w/o pain) groups, and each exercise was performed for eight weeks. TMDs pain intensity was assessed using a 100-mm visual analog scale (VAS), and the range-of-mouth opening was evaluated at the baseline (T0), 2 weeks (T1), 4 weeks (T2), and 8 weeks (T3). Of the 61 participants, 57 (JE w/pain group, n = 30; JE w/o pain group, n = 27) were included in the analysis. The range-of-mouth opening and TMDs pain intensity improved from T1 to T3 in both groups. The JE w/pain group showed significant differences at T3 compared to T1 (pain-free unassisted mouth opening, p = 0.006; jaw-opening pain, p = 0.014; chewing pain, p = 0.018). In addition, the JE w/pain group showed significantly greater changes in the maximum unassisted mouth opening at T2 and T3 than the JE w/o pain group (T2, p < 0.001; T3, p = 0.003). Thus, jaw-opening exercises, until the occurrence of pain, may be effective in patients with TMDs.

The gap between the Markov blanket and ontological boundaries arises from the former's inability to capture the dynamic process through which biological and cognitive agents actively generate their own boundaries with the environment. Active inference in the free-energy principle (FEP) framework presupposes the existence of a Markov blanket, but it is not a process that actively generates the latter.

Transforming growth factor β (TGF-β) increases epithelial cancer cell migration and metastasis by inducing epithelial-mesenchymal transition (EMT). TGF-β also inhibits cell proliferation by inducing G1 phase cell-cycle arrest. However, the correlation between these tumor-promoting and -suppressing effects remains unclear. Here, we show that TGF-β confers higher motility and metastatic ability to oral cancer cells in G1 phase. Mechanistically, keratin-associated protein 2-3 (KRTAP2-3) is a regulator of these dual effects of TGF-β, and its expression is correlated with tumor progression in patients with head and neck cancer and migratory and metastatic potentials of oral cancer cells. Furthermore, single-cell RNA sequencing reveals that TGF-β generates two populations of mesenchymal cancer cells with differential cell-cycle status through two distinctive EMT pathways mediated by Slug/HMGA2 and KRTAP2-3. Thus, TGF-β-induced KRTAP2-3 orchestrates cancer cell proliferation and migration by inducing EMT, suggesting motile cancer cells arrested in G1 phase as a target to suppress metastasis., Competing Interests: Declaration of interests The authors do have a patent related to this work (patent application number [Japan Patent Office]: P2021-214914)., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The blood and lymphatic vasculature networks are not yet fully understood even in mouse because of the inherent limitations of imaging systems and quantification methods. This study aims to evaluate the usefulness of the tissue-clearing technology for visualizing blood and lymphatic vessels in adult mouse. Clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) enables us to capture the high-resolution 3D images of organ- or area-specific vascular structures. To evaluate these 3D structural images, signals are first classified from the original captured images by machine learning at pixel base. Then, these classified target signals are subjected to topological data analysis and non-homogeneous Poisson process model to extract geometric features. Consequently, the structural difference of vasculatures is successfully evaluated in mouse disease models. In conclusion, this study demonstrates the utility of CUBIC for analysis of vascular structures and presents its feasibility as an analysis modality in combination with 3D images and mathematical frameworks., (© 2022. The Author(s).)

I would like to congratulate the Journal of Biochemistry (JB) on its 100th anniversary. I served as the Editor-in-Chief for four years starting in 2010 and succeeded Dr Naoyuki Taniguchi., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Transforming growth factor β (TGFβ) induces epithelial-mesenchymal transition (EMT), which correlates with stemness and invasiveness. Mesenchymal-epithelial transition (MET) is induced by TGFβ withdrawal and correlates with metastatic colonization. Whether TGFβ promotes stemness and invasiveness simultaneously via EMT remains unclear. We established a breast cancer cell model expressing red fluorescent protein (RFP) under the E-cadherin promoter. In 2D cultures, TGFβ induced EMT, generating RFP low cells with a mesenchymal transcriptome, and regained RFP, with an epithelial transcriptome, after MET induced by TGFβ withdrawal. RFP low cells generated robust mammospheres, with epithelio-mesenchymal cell surface features. Mammospheres that were forced to adhere generated migratory cells, devoid of RFP, a phenotype which was inhibited by a TGFβ receptor kinase inhibitor. Further stimulation of RFP low mammospheres with TGFβ suppressed the generation of motile cells, but enhanced mammosphere growth. Accordingly, mammary fat-pad-transplanted mammospheres, in the absence of exogenous TGFβ treatment, established lung metastases with evident MET (RFP high cells). In contrast, TGFβ-treated mammospheres revealed high tumour-initiating capacity, but limited metastatic potential. Thus, the biological context of partial EMT and MET allows TGFβ to differentiate between pro-stemness and pro-invasive phenotypes., (© 2022 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-β (TGF-β) family, are multifunctional cytokines. BMPs have a broad range of functions, and abnormalities in BMP signaling pathways are involved in cancer progression. BMPs activate the proliferation of certain cancer cells. Malignant phenotypes of cancer cells, such as increased motility, invasiveness, and stemness, are enhanced by BMPs. Simultaneously, BMPs act on various cellular components and regulate angiogenesis in the tumor microenvironment. Thus, BMPs function as pro-tumorigenic factors in various types of cancer. However, similar to TGF-β, which shows both positive and negative effects on tumorigenesis, BMPs also act as tumor suppressors in other types of cancers. In this article, we review important findings published in the recent decade and summarize the pro-oncogenic functions of BMPs and their underlying mechanisms. The current status of BMP-targeted therapies for cancers is also discussed., Competing Interests: SE and KM were partly supported by Eisai, Co., Ltd., (Copyright © 2022 Ehata and Miyazono.)

We developed an in vivo serial passaging model for renal cancer with orthotopic renal subcapsular inoculation. We detail the procedures for renal subcapsular inoculation of cancer cells in mice, followed by in vivo and ex vivo bioluminescence imaging, tumor-bearing kidney dissociation, and in vivo passaging. This protocol is capable of reproducing the coevolution between cancer cells and the primary tumor microenvironment. It enables us to unveil the systemic dynamics of metastasis and develop a therapeutic strategy for metastatic renal cancer. For complete details on the use and execution of this protocol, please refer to Nishida et al. (2020)., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Smad proteins transduce signals downstream of transforming growth factor-β (TGF-β) and are one of the factors that regulate the expression of genes related to diseases affecting the skin. In the present study, we identified MAB21L4, also known as male abnormal 21 like 4 or C2orf54, as the most up-regulated targets of TGF-β and Smad3 in differentiated human progenitor epidermal keratinocytes using chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq). We found that TGF-β induced expression of the barrier protein involucrin (encoded by the IVL gene). Transcriptional activity of the IVL promoter induced by TGF-β was inhibited by MAB21L4 siRNAs. Further analysis revealed that MAB21L4 siRNAs also down-regulated the expression of several target genes of TGF-β. MAB21L4 protein was located mainly in the cytosol, where it was physically bound to Smad3 and a transcriptional corepressor c-Ski. siRNAs for MAB21L4 did not inhibit the binding of Smad3 to their target genomic regions but down-regulated the acetylation of histone H3 lys 27 (H3K27ac), an active histone mark, near the Smad3 binding regions. These findings suggest that TGF-β-induced MAB21L4 up-regulates the gene expression induced by TGF-β, possibly through the inhibition of c-Ski via physical interaction in the cytosol., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society.)

The activation of RIG-I-like receptor (RLR) signaling in cancer cells is widely recognized as a critical cancer therapy method. The expected mechanism of RLR ligand-mediated cancer therapy involves the promotion of cancer cell death and strong induction of interferon (IFN)-β that affects the tumor microenvironment. We have recently shown that activation of RLR signaling in triple-negative breast cancer cells (TNBC) attenuates transforming growth factor-β (TGF-β) signaling, which partly contributes to the promotion of cancer cell pyroptosis. However, the consequences of suppression of TGF-β signaling by RLR ligands with respect to IFN-β-mediated tumor suppression are not well characterized. This study showed that transfection of a typical RLR ligand polyI:C in cancer cells produces significant levels of IFN-β, which inhibits the growth of the surrounding cancer cells. In addition, IFN-β-induced cell cycle arrest in surrounding cancer cells was inhibited by the expression of constitutively active Smad3. Constitutively active Smad3 suppresses IFN-β expression through the alleviation of IFN regulatory factor 3 binding to the canonical target genes, as suggested by ChIP sequencing analysis. Based on these findings, a new facet of the protumorigenic function of TGF-β that suppresses IFN-β expression is suggested when RLR-mediated cancer treatment is used in TNBC., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

DNA hypermethylation is frequently observed in clear cell renal cell carcinoma (ccRCC) and correlates with poor clinical outcomes. However, the detailed function of DNA hypermethylation in ccRCC has not been fully uncovered. Here, we show the role of DNA methylation in ccRCC progression through the identification of a target(s) of DNA methyltransferases (DNMT). Our preclinical model of ccRCC using the serial orthotopic inoculation model showed the upregulation of DNMT3B in advanced ccRCC. Pretreatment of advanced ccRCC cells with 5-aza-deoxycytidine, a DNMT inhibitor, attenuated the formation of primary tumors through the induction of apoptosis. DNA methylated sites were analyzed genome-wide using methylation array in reference to RNA-sequencing data. The gene encoding ubiquinol cytochrome c reductase hinge protein (UQCRH), one of the components of mitochondrial complex III, was extracted as a methylation target in advanced ccRCC. Immunohistochemical analysis revealed that the expression of UQCRH in human ccRCC tissues was lower than normal adjacent tissues. Silencing of UQCRH attenuated the cytochrome c release in response to apoptotic stimuli and resulted in enhancement of primary tumor formation in vivo, implying the tumor-suppressive role of UQCRH. Moreover, 5-aza-deoxycytidine enhanced the therapeutic efficiency of mammalian target of rapamycin inhibitor everolimus in vivo. These findings suggested that the DNMT3B-induced methylation of UQCRH may contribute to renal cancer progression and implicated clinical significance of DNMT inhibitor as a therapeutic option for ccRCC., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Glioma-initiating cells (GICs), a major source of glioblastoma recurrence, are characterized by the expression of neural stem cell markers and the ability to grow by forming nonadherent spheres under serum-free conditions. Bone morphogenetic proteins (BMPs), members of the transforming growth factor-β family, induce differentiation of GICs and suppress their tumorigenicity. However, the mechanisms underlying the BMP-induced loss of GIC stemness have not been fully elucidated. Here, we show that paired related homeobox 1 (PRRX1) induced by BMPs decreases the CD133-positive GIC population and inhibits tumorigenic activity of GICs in vivo. Of the two splice isoforms of PRRX1, the longer isoform, pmx-1b, but not the shorter isoform, pmx-1a, induces GIC differentiation. Upon BMP stimulation, pmx-1b interacts with the DNA methyltransferase DNMT3A and induces promoter methylation of the PROM1 gene encoding CD133. Silencing DNMT3A maintains PROM1 expression and increases the CD133-positive GIC population. Thus, pmx-1b promotes loss of stem cell-like properties of GICs through region-specific epigenetic regulation of CD133 expression by recruiting DNMT3A, which is associated with decreased tumorigenicity of GICs., (© 2021 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

This paper investigates the role of group identification in empathic emotion and its behavioral consequences. Our central idea is that group identification is the key to understanding the process in which empathic emotion causes helping behavior. Empathic emotion causes helping behavior because it involves group identification, which motivates helping behavior toward other members. This paper focuses on a hypothesis, which we call "self-other merging hypothesis (SMH)," according to which empathy-induced helping behavior is due to the "merging" between the helping agent and the helped agent. We argue that SMH should be interpreted in terms of group identification. The group identification interpretation of SMH is both behaviorally adequate (i.e., successfully predicts and explains the helping behavior in the experimental settings) and psychologically plausible (i.e., does not posit psychologically unrealistic beliefs, desires, etc.). Empathy-induced helping behavior, according to the group identification interpretation of the SMH, does not fit comfortably into the traditional egoism/altruism dichotomy. We thus propose a new taxonomy according to which empathy-induced helping behavior is both altruistic at the individual level and egoistic at the group level., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a past co-authorship with one of the author KM., (Copyright © 2021 Miyazono and Inarimori.)

We previously reported that bone morphogenetic protein (BMP) signaling promotes tumorigenesis in gynecologic cancer cells. BMP2 enhances proliferation of ovarian and endometrial cancer cells via c-KIT induction, and triggers epithelial-mesenchymal transition (EMT) by SNAIL and/or SLUG induction, leading to increased cell migration. However, the downstream effectors of BMP signaling in gynecological cancer cells have not been clearly elucidated. In this study, we performed RNA-sequencing of Ishikawa endometrial and SKOV3 ovarian cancer cells after BMP2 stimulation, and identified TNFRSF12A, encoding fibroblast growth factor-inducible 14 (FN14) as a common BMP2-induced gene. FN14 knockdown suppressed BMP2-induced cell proliferation and migration, confirmed by MTS and scratch assays, respectively. In addition, FN14 silencing augmented chemosensitivity of SKOV3 cells. As a downstream effector of BMP signaling, FN14 modulated both c-KIT and SNAIL expression, which are important for growth and migration of ovarian and endometrial cancer cells. These results support the notion that the tumor promoting effects of BMP signaling in gynecological cancers are partially attributed to FN14 induction., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Follistatin-like 3 (FSTL3) is an endogenous antagonist against transforming growth factor-β family ligands. Monovalent FSTL3-Fc fusion protein (mono-FSTL3-Fc) generated with knobs-into-holes technology overcomes limitations of current anti-myostatin therapies. We have developed a facile protocol for affinity purification of the Fc-fused protein from the supernatant of HEK293T cells stably expressing the protein. This protocol is advantageous by only requiring readily accessible equipment. We further outline the steps for validation of mono-FSTL3-Fc increasing systemic muscle mass in mice after intraperitoneal administration. For complete details on the use and execution of this protocol, please refer to Ozawa et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)