{"paper_id":"382f3bdd-7080-484b-aec5-d61f0e2a7da0","body_text":"1Scientific  RepoRtS  |         (2019) 9:12094  | https://doi.org/10.1038/s41598-019-48580-9\nwww.nature.com/scientificreports\nMicrogravity inhibits \ndecidualization via decreasing Akt \nactivity and FOXO3a expression in \nhuman endometrial stromal cells\nHye-Jeong cho 1,2,3, Mi-Ock Baek1,2,3, Sana Abdul Khaliq1,2,3, Seung Joo chon 4, Kuk Hui Son5, \nSung Ho Lee6 & Mee-Sup Yoon  1,2,3\nDecidualization is characterized by the differentiation of endometrial stromal cells (eSCs), which is \ncritical for embryo implantation and maintenance of pregnancy. In the present study, we investigated \nthe possible effect of simulated microgravity (SM) on the process of proliferation and in vitro \ndecidualization using primary human eSCs. Exposure to SM for 36 h decreased the proliferation and \nmigration of eSCs significantly, without inducing cell death and changes in cell cycle progression. The \nphosphorylation of Akt decreased under SM conditions in human eSCs, accompanied by a simultaneous \ndecrease in the level of matrix metalloproteinase (MMP)-2 and FOXO3a. Treatment with Akti, an Akt \ninhibitor, decreased MMP-2 expression, but not FOXO3a expression. The decreased level of FOXO3a \nunder SM conditions impeded autophagic flux by reducing the levels of autophagy-related genes. \nIn addition, pre-exposure of eSCs to SM significantly inhibited 8-Br-cAMP induced decidualization, \nwhereas restoration of the growth status under SM conditions by removing 8-Br-cAMP remained \nunchanged. Treatment of human eSCs with SC-79, an Akt activator, restored the reduced migration of \neSCs and decidualization under SM conditions. In conclusion, exposure to SM inhibited decidualization \nin eSCs by decreasing proliferation and migration through Akt/MMP and FOXO3a/autophagic flux.\nHuman space exploration has been growing in recent years. This has led researchers to investigate the effect of \nharsh environmental conditions, including extreme temperature, ionizing radiation, and altered gravity. Among \nthem, exposure to microgravity has been reported to result in various detrimental effects on the muscular mass\n1, \nimmune system2, cardiovascular system 3, bone mass1, nervous system 4, and endocrine system 5. Conditions of \nnear weightlessness affects cell growth, differentiation, apoptosis, and autophagy in different cells6. However, the \neffect of microgravity on embryo implantation and maintenance of pregnancy in the human endometrium has \nnot been examined yet.\nDecidualization, characterized by the differentiation of endometrial stromal cells, is a profound change in \nthe cells of the endometrium for embryo implantation and maintenance of pregnancy\n7. During the process of \ndecidualization, the fibroblast-like endometrial stromal cells (eSCs) acquire a round shape, with accumulation of \nglycogen and lipids, secretion of growth factors and cytokines, such as prolactin (PRL) and insulin-like growth \nfactor binding protein 1 (IGFBP1), and accumulation of extracellular matrix (ECM). Although progesterone/\ncAMP are well known inducers of decidualization, mechanical stretch in the endometrium has been reported to \ninduce decidualization by regulating the expression of IGFBP-1\n7. In addition, mechanical stretch is transduced \ninto biochemical signals via interleukin (IL)-8 to regulate endometrial differentiation8. Subendometrial myome-\ntrial contraction also has been shown to occur throughout the menstrual cycle, which is related to other uterine \n1Department of Molecular Medicine, School of Medicine, Gachon University, Incheon, 21999, Republic of Korea. \n2Lee Gil Ya Cancer and Diabetes Institute, Gachon University, Incheon, 21999, Republic of Korea. 3Department of \nHealth Sciences and Technology, GAIHST, Gachon University, Incheon, 21999, Republic of Korea. 4Department of \nObstetrics and Gynecology, Gachon University Gil Medical Center, College of Medicine, Gachon University, Incheon, \n21565, Republic of Korea. 5Department of Thoracic and Cardiovascular Surgery, Gachon University Gil Medical \nCenter, College of Medicine, Gachon University, Incheon, 21565, Republic of Korea. 6Department of Thoracic \nand Cardiovascular Surgery, Korea University, Seoul, 02841, Republic of Korea. Correspondence and requests for \nmaterials should be addressed to M.-S. Y . (email: msyoon@gachon.ac.kr)\nReceived: 8 April 2019\nAccepted: 8 August 2019\nPublished: xx xx xxxx\nopen\n\n\n2Scientific  RepoRtS  |         (2019) 9:12094  | https://doi.org/10.1038/s41598-019-48580-9\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nmovements, such as endometriosis and mentruation 9. These findings led us to investigate the possible effect of \nmicrogravity on the proliferation and differentiation of eSCs.\nIn the current study, we hypothesize that simulated microgravity (SM) is a critical regulator of the deciduali-\nzation of human eSCs. To verify this hypothesis, we examined the changes in the proliferation and migration of \nSM-exposed eSCs and analyzed the effects of decidualization exposure to SM before the initiation of deciduali-\nzation. Taken together, we suggested that mechanical loading is a critical factor in the regulation of proliferation \nand decidualization in human eSCs, proposing an airspace strategy to protect our body.\nResults\nSM inhibits the growth of primary human eSCs without cell death. We first examined whether a \nreduced gravitational force affects the growth rate of primary human eSCs using a clinostat, a device that is widely \nused for generating SM. Primary eSCs grown to 80–90% confluency were placed in either a dynamic reactor to \nsimulate 0 g or in a stationary control (1 g). After rotation of the reactor in both vertical and horizontal planes, the \nnumber of eSCs was measured at 0, 12, 24, and 36  h. Human eSCs grew at a significantly slower rate under SM \nconditions (30 and 17% reduction was seen at 24 and 36 h, respectively) compared to those grown under terres-\ntrial gravity (Fig. 1A). The percentage of dead cells under SM conditions remained unchanged compared to those \nunder conditions of terrestrial gravity, as indicated by the number of both 7-aminoactinomycin D (7-AAD)\n+ and \npropidium iodine (PI)+ cells (Fig. 1B), suggesting no difference in viability between either condition. Consistent \nwith these findings, the level of Ki-67 in SM-exposed eSCs was mildly decreased, but not significantly, compared \nto that of a stationary control (Fig.  1C,D). As shown in Fig.  1E, the ratios of cells in the G1, G2, and S phases \nunder SM conditions were comparable to those of cells under terrestrial gravity, indicating no changes in the pro-\ngression of cell cycle under SM conditions. These results indicate that exposure to SM reduces the proliferation of \neSCs without a discernible increase in the apoptotic cell fraction or changes in cell cycle progression.\nSM inhibits the migration of primary human eSCs. Migration of human eSCs is required for human \nembryonic trophoblast invasion10. We analyzed the effect of SM on the migration of primary eSCs using a wound \nhealing scratch assay. After 0, 6, 12, and 24 h under SM conditions, we examined cell motility by assessing changes \nin both cell-free area and the number of migrated cells. Exposure of eSCs to SM caused a significant decrease in \ncell motility of the stained image of cells (Fig. 2A), increase in the remaining cell-free area (Fig. 2B), and decrease \nin the number of migrated cells (Fig. 2C). The remaining cell-free area and number of migrated cells at 12 h after \nSM exposure were increased by 1.6-fold and decreased by 3.7-fold, respectively, as compared to exposure to 1 g, \nsuggesting that eSCs under SM migrate evidently slower (Fig. 2B,C). Consistent with this observation, the level \nof MMP-2 and MMP-9, well-known regulators that degrade the ECM, and the phosphorylation of β-catenin, a \nFigure 1. SM inhibits the growth of human eSCs without any changes in cell death and cell cycle progression. \n(A) Primary human eSCs were incubated either under terrestrial gravity (1 g) or under SM for indicated \ntime periods and counted using a cell counter. (B) The cells were treated as (A) for 36 h, stained with either \n7-AAD or PI, and then analyzed by flow cytometry. (C) The cells were treated as in (B), lysed, and analyzed \nby western blotting. (D) Western blot images were analyzed using ImageJ to determine the relative protein \nexpression of Ki-67 (using tubulin as the internal control). (E) The cells were treated as (B), stained with PI and \nanalyzed using flow cytometry. Abbreviations: simulated microgravity (SM); 7-aminoactinomycin D (7-AAD); \npropidium iodine (PI). Data are expressed as mean ± standard deviation (SD), with paired t-tests performed as \nindicated. *P < 0.05, **P < 0.01 versus control at each indicated time.\n\n3Scientific  RepoRtS  |         (2019) 9:12094  | https://doi.org/10.1038/s41598-019-48580-9\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\ncomponent of cell to cell connection11, were decreased in eSCs exposed to SM for 36 h (Fig. 2D,E), thereby con-\nfirming that exposure to SM leads to slow migration of eSCs.\nSM reduces Akt activity in human eSCs. Akt is critical to the regulation of cell migration and growth12. \nNext, we examined the phosphorylation of Akt to assess the activity of Akt. Akt phosphorylation at both serine \n473 and threonine 308 decreased significantly (up to 88 and 85%, respectively) in eSCs exposed to SM for 36 h \n(Fig. 3A) compared to that in cells under 1 g (Fig. 3B). Akt promotes cell growth via translational regulation by \nactivating the mTOR complex1 (mTORC1) 13. However, the phosphorylation of S6K1 (at threonine 389) and \neukaryotic initiation factor 4E binding protein 1 (4EBP1) (at serine 65 and at threonine 37 and 46), which are \nwell known downstream targets of mTORC1, was not changed significantly (Fig.  3A,B). Exposure to SM for \n36 h did not affect the expression of mTOR, raptor, and rictor, which are the major components of mTORC1 \nand mTORC2 (Fig.  3C,D). In addition, the level of either raptor or rictor in the mTOR complexes remained \nunchanged (Fig. 3E). Interestingly, the phosphorylation of NDRG, a downstream target of SGK that is regulated \nby mTORC2, also remained unchanged under SM conditions (Fig. 3E), indicating that the decrease in Akt phos-\nphorylation did not originate from the inhibition of mTORC2 activity. Next, to examine the involvement of Akt \nin the growth and migration of cells, we treated eSCs with Akti, a selective inhibitor of Akt, for the indicated time \nperiods. The growth of eSCs decreased (Fig. 4A) by 23% at 24 h and 25% at 36 h, with no significant changes in cell \ndeath and cell cycle progression, as evidenced by the frequencies of 7-AAD\n+ and PI+ cells (Fig. 4B) and the ratios \nof cells in the G1, G2, and S phases (Fig. 4C), respectively. In addition, the migration of eSCs was reduced in the \npresence of Akti, as shown by the stained images of eSCs with reduced motility (Fig. 4D), the increased free area \nbetween cells (Fig.  4E), and the deceased number of migrated cells (Fig.  4F). Treatment with Akti significantly \nreduced the expression level of MMP-2. However, the phosphorylation of β-catenin remained unchanged in the \npresence of Akti (Fig.  4G,H), indicating that reduction of β -catenin phosphorylation might be involved in the \ndecrease of cell motility in an Akt-independent manner. Insulin induced the phosphorylation of Akt, but not \nβ-catenin in human eSCs (SFig. 1), supporting the Akt-independent regulation of β -catenin phosphorylation. \nMoreover, treatment with SC-79, an Akt activator, restored the reduced migration of human eSCs under SM \nconditions (Fig. 4I,J,K), confirming that Akt regulates the migration of eSCs under SM conditions. These results \nsuggested that exposure to SM inhibited the growth and migration of eSCs through inactivation of Akt, resulting \nin a decrease of MMP-2 expression.\nSM suppresses FOXO3a protein expression. FOXO3a regulates the transcription of MMPs in decidual-\nized human eSCs14. In order to investigate the involvement of FOXO3a in SM-induced migration through control \nof MMP-2 expression, we next examined the expression and phosphorylation of FOXO3a under SM conditions. \n0\n20\n40\n60\n80\n100\n120\nChange of\n cell free area ( %) **\n**\n06 12 24 (hour)\n+SM- SM\nB\n- SM\n+SM\n06 12 24 (hour)\nA\nC\nMigrated cell no.(%)\nD\n-+SM\nMMP2\nMMP9\npS33/37/T41-\nβ-catenin\nβ-catenin\ntubulin\n+SM- SM\npβ-catenin/\nβ-catenin\n0.0\n0.2\n0.4\n0.6\n0.8\n1.0\n1.2\n noitalyrohpsohp evitaleR\nnoisserpxe evitaleR/\nMMP2 MMP9\n****\nE\n0\n20\n40\n60\n80\n100\n120\n+SM\n- SM\n06 12 24 (hour)\n**\n**\nFigure 2. SM impedes the migration of human eSCs. (A–C) Human eSCs were scratched with a T200 tip and \nthen incubated under 1 g or SM for the indicated times. (A) Cells were stained using the CytoPainter Cell Tracking \nStaining Kit and photographed. (B) The cell-free area was measured using ImageJ and change of cell-free area was \ncalculated. (C) The number of migrated cells was counted using ImageJ. (D) The cells were incubated either under \nterrestrial gravity (1 g) or under SM for 36 h, lysed, and analyzed by western blotting. (E) Western blot images \nwere analyzed using ImageJ to determine the relative protein expression of MMP-2 and MMP-9 (using tubulin \nas the internal control) and the relative phosphorylation of S33/37/T41-β-catenin (using β-catenin as the internal \ncontrol). Abbreviations: simulated microgravity (SM). Data are expressed as mean ± SD, with paired t-tests \nperformed as indicated. **P < 0.01, *P < 0.05 versus control at each indicated time.\n\n4Scientific  RepoRtS  |         (2019) 9:12094  | https://doi.org/10.1038/s41598-019-48580-9\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nAkt phosphorylates FOXOs to inhibit their transcriptional activity by exporting FOXOs from the nucleus15. The \nlevel and phosphorylation of FOXO3a decreased in eSCs exposed to SM for 36 h (Fig. 5A,B). The phosphoryl-\nation and level of FOXO1 remained unchanged (Fig. 5C,D), suggesting that decrease in FOXO3a expression is \nnot a common phenotype in FOXOs under SM conditions. FOXO3a is involved in the transcriptional regulation \nof proteins in diverse cellular pathways, including cell cycle inhibition, autophagy, and apoptosis 15. No signif-\nicant reduction in the level of caspase-3 and cleaved caspase-3, a central regulator of apoptosis, was observed \n(Fig.  5E,F), and the population of apoptotic cells remained unchanged in eSCs under SM conditions com-\npared to that in cells under 1 g condition, as shown by fluorescence-activated cell sorting (FACS) analysis using \nannexin-V/propidium iodine (PI) double staining (Fig.  5G). Exposure of eSCs to SM decreased the expression \nof autophagy-related regulators, including Vps15, beclin1, and UVrag (Fig. 5H,I). The level of LC3BII, the repre-\nsentative marker of autophagic flux, decreased, indicating a decrease in autophagic flux (Fig. 5J,K), which agreed \nwith the decrease in autophagic gene expression. The level of p62 protein decreased in eSCs under SM conditions \n(SFig. 2B,C), due to a reduction in p62 mRNA expression (SFig. 2A). Consistent with these results, treatment with \n3-methyladenine (3-MA), an inhibitor of autophagy that inhibits Vps34, decreased the growth of eSCs (Fig. 5L) \nas well as the migration of eSCs, as indicated by the images of migrated cells (Fig.  5M), change of cell-free area \n(Fig. 5N), and number of migrated cells (Fig. 5O). In addition, co-treatment of eSCs with 3-MA and Akti resulted \nAkt\npT389-S6K1\npS65-4EBP1\nSM :\ntubulin\npT37/46-4EBP1\n4EBP1\n-+\nS6K1\nA\n-+SM:\nraptor\nmTOR\nrictor\ntubulin\nB\n-+SM : -+\n-SM +SM\n**\n evitaleR\n noitalyrohpsohp\nC\nmTOR: IPlysate\nraptor\nmTOR\nAkt\nrictor\npS473-Akt\ntubulin\npT346-NDRG\nNDRG\nmTOR raptor rictor\n0.0\n0.2\n0.4\n0.6\n0.8\n1.0\n1.2 -SM +SMnoisserpxe evitaleR\npT308-Akt\nD E\n0.0\n0.2\n0.4\n0.6\n0.8\n1.0\n1.2\npS473-Ak\nt\npT308-Ak\nt\npS65-4EBP1\npT37/46-4EBP\n1\npT389-S6K1\n*\npS473-Akt\nFigure 3. SM decreased Akt activity in human eSCs. (A–D) Human eSCs were incubated either under \nterrestrial gravity (1 g) or under SM for 36 h, lysed, and analyzed by western blotting (A,C). Western blot images \nwere analyzed using ImageJ to determine the phosphorylation of pS473-Akt relative to Akt, pT308-Akt relative \nto Akt, pT389-S6K1 relative to S6K1, pS65-4EBP1 relative to 4EBP1, and pT47/36-4EBP1 relative to 4EBP1 (B), \nand the expression of mTOR, raptor, and rictor relative to tubulin (D). The cells were treated as in (A), subjected \nto immunoprecipitation using antibodies against mTOR, and analyzed by western blotting. Abbreviations: \nsimulated microgravity (SM). Data are expressed as mean ± SD, with paired t-tests performed as indicated. \n*P < 0.05, **P < 0.01 versus control at each indicated time.\n\n5Scientific  RepoRtS  |         (2019) 9:12094  | https://doi.org/10.1038/s41598-019-48580-9\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nin the highly reduced proliferation (Fig.  6A) and migration of eSCs (Fig.  6B,C,D). These results indicate that a \ndecrease in Akt activity and autophagic flux may induce slow migration under SM conditions. However, FOXO3a \nlevel remained unchanged after treatment with Akti, although FOXO3a phosphorylation decreased (SFig. 3), \nsuggesting that FOXO3a expression level under SM conditions is regulated in an Akt-independent manner.\nSM suppresses the decidualization of primary human eSCs. Akt and FOXO3a are critical regulators \nof decidualization of eSCs 14. Next, we proceeded to test whether SM affects the efficiency of decidualization in \neSCs. First, we pre-exposed the cells to either SM or 1 g conditions for one day and then induced decidualization \nby shifting to differentiation medium with 0.5 mM 8-Br-cAMP for one day. Pre-exposure to SM significantly \nsuppressed decidualization, which was indicated by a decrease in the mRNA expression of PRL and IGFBP1 \n(Fig. 7A), decidua-like morphological changes (Fig.  7B), and senescence-associated β -galactosidase (SAβ G)\n+ \ncells, which were reported to increase during decidualization16 (Fig. 7C), suggesting that pretreatment with SM \nresults in defective decidualization. When eSCs were induced for decidualization under SM conditions for one \nday, the mRNA levels of PRL and IGFBP1 were reduced (Fig. 7D). In addition, exposure to SM for one day after \nthe induction of decidualization by adding 8-Br-cAMP inhibited the mRNA expression of PRL and IGFBP1 \n(Fig. 7E). However, when decidualized human eSCs were restored to an undifferentiated phenotype upon with-\ndrawal of 8-Br-cAMP , as previously reported\n17, the reverse process of decidualization under SM condition was \ncomparable to that under 1 g condition (Fig. 7F), indicating no effect of SM exposure on restoration of growth \nstatus in human eSCs. When the eSCs were pre-exposed to SM in the presence of SC-79 for one day and induced \nto differentiation by the addition 8-Br-cAMP , the mRNA expression of PRL and IGFBP1 was partially restored \nBA\n- +Akti\nMMP2\ntubulin\npS33/37/T41-\nβ-catenin\nβ-catenin\n- Akti\n+Akti\n06 12 24 (hour)\n06 12 24\n0\n20\n40\n60\n80\n100\n120\nChange of\n cell free area ( %)\nD\nGE\n0.0\n0.2\n0.4\n0.6\n0.8\n1.0\n1.2\n1.4\n1.6\n*\n0\n100\n200\n300\n400\n500\n*\n*- Akti\n+Akti\n01 22 43 6( hour)\n(hour)\nCell proliferation ( %)\npβ\n-caten\nin/\nβ-cate\nnin\n noitalyrohpsohp evitaleR\nnoisserpxe evitaleR/\nMMP2\n- Akti\n+Akti\nH\n**\n** **\nC\n% gated cells\nG0/G1 S G2/M0\n20\n40\n60\n80\n100\nMigrated cell no.(%)\nF\n0\n20\n40\n60\n80\n100\n120\n140\n06 12 24 (hour)\n- Akti\n+Akti\n*\n**\n*\n0\n1\n2\n3\n4\n5\n6\nPI+\n7-AAD+\nFrequency ( %)\n-Akt-i +Akt-i\n- Akti\n+Akti\n- Akti\n+Akti\n0\n20\n40\n60\n80\n100\n120\nChange of \nCell free area ( %)\nJ - SM\n+SM\n+SM+SC-79\n**\n02 4 (hour)\nK\nMigrated cell no.(%)24 hours\n**\n0\n20\n40\n60\n80\n100\n120\n- SM\n+SM\n+SM+SC-79\n24 hours\n0h\nI\n- SM\n- SM\n+SM\n+SM+SC-79\nFigure 4. Akt decreased cell growth and migration in human eSCs. (A) Human eSCs were incubated with or \nwithout 1 μM Akti for the indicated times. The cells were counted using a cell counter. (B) The cells were treated \nas in (A), stained with either 7-AAD or PI, and analyzed by flow cytometry. (C) The cells were treated as in \n(A), stained with PI, and analyzed by flow cytometry. (D–F) The cells were scratched with a T200 tip and then \nincubated with or without 1 μM Akti for the indicated times. (D) The cells were stained using the CytoPainter \nCell Tracking Staining Kit and photographed. (E) The cell-free area was measured using ImageJ and change of \ncell-free area was calculated. (F) The number of migrated cells was counted using ImageJ. (G,H) The cells were \ntreated as in (A), lysed, and subjected to western blotting. (H) Western blot images were analyzed using ImageJ \nto determine the phosphorylation of pS33/37/T41-β-catenin relative to β-catenin and the expression of MMP-2 \nrelative to tubulin. (I–K) The cells were scratched with a T200 tip and incubated with or without 0.2 μg/ml \nSC-79 under SM conditions for 24 h. (I) The cells were stained using the CytoPainter Cell Tracking Staining kit \nand photographed. (J) Cell-free areas were measured using ImageJ and the changes in the cell-free areas were \ncalculated. (K) The number of migrated cells was counted using ImageJ. Abbreviations: simulated microgravity \n(SM); 7-aminoactinomycin D (7-AAD); propidium iodine (PI). Data are expressed as mean ± SD, with paired \nt-tests performed as indicated. *P < 0.05, **P < 0.01 versus control at each indicated time; °°P < 0.01 versus SM \nexposed cells.\n\n6Scientific  RepoRtS  |         (2019) 9:12094  | https://doi.org/10.1038/s41598-019-48580-9\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\n(Fig. 7G), suggesting that the inhibition of Akt under SM conditions resulted in decidualization defects. These \nresults suggested that exposure to SM reduced decidualization specifically in eSCs.\nDiscussion\nDecidualization is required to facilitate implantation and maintain pregnancy 18. Subendometrial myometrial \nmovement in the endometrium induces biochemical signals, which trigger endometrial physiological and patho-\nlogical responses9. Although mechanical loading has been shown to promote decidualization7, the mechanism by \nwhich it controls the differentiation of human eSCs is unclear. Here, we showed that exposure to SM (mechanical \nunloading by a clinostat) inhibited decidualization in eSCs by modulating their proliferation and migration. \nExposure to SM decreased Akt activity and FOXO3a expression, leading to suppression of MMP-2 expression \nand autophagic flux, respectively. Hence, we propose that mechanical unloading inhibits decidualization through \ninhibiting Akt- and FOXO3a-dependent cell growth and migration (Fig. 8).\nInhibition of decidualization under SM condition suggested that mechanical unloading plays a role in the dif-\nferentiation of eSCs. We found that exposure to SM suppressed decidualization during both the initial and middle \nstages of decidualization (Fig. 7D,E). Mechanical loading enhances decidualization through stimulation of IL-8 \nsecretion\n8, and the production and secretion of IGFBP17. Notably, when the cells were pre-exposed to SM before \ninitiation of decidualization, the expression of the decidualization markers, PRL and IGFBP1, and morphological \nchange were significantly decreased (Fig. 7A,B), indicating that changes in the growth rate of eSCs could induce \nprofound effects on the cells, resulting in defective decidualization. In agreement with our observation, the effi-\nciency of decidualization is determined by both the growth rate and the extent of migration of eSCs\n19.\nAkt activity directly and indirectly regulates cell migration by regulating the actin cytoskeleton 20, cell-cell \nadhesion, cell motility, and extracellular degradation12. Decreased Akt activity induces mesenchymal to epithelial \ntransition (MET), a counterpart of epithelial to mesenchymal transition, which increases cell-cell adhesion and \nreduces cell motility\n21. Induction of decidualization also inhibits Akt activity, resulting in partial induction of \nMET-like molecular changes21. In the current study, exposure to SM decreased cell migration through regulation \nof Akt activity (Fig.  4I,J,K), resulting in the inhibition of decidualization during the initial and middle stages \nFigure 5. FOXO3a expression and autophagic flux decreased under SM condition in human eSCs. (A–F,H–K) \nHuman eSCs were incubated either under terrestrial gravity (1 g) or under SM for 36 h, lysed, and subjected \nto western blotting. (B,D,F,I,K) ImageJ was used to analyze the following: the expression level of FOXO3a \nrelative to tubulin (B), the phosphorylation of pS256-FOXO1 relative to FOXO1 (D), the expression level of \ncaspase-3 and cleaved caspase-3 relative to tubulin (F), the expression level of Vps34, Vps15, Atg14L, beclin1, \nand UVrag relative to tubulin (I), and the expression of LC3BII relative to tubulin (K). (G) Cells were treated as \nin (A), stained with PI and annexin V , and analyzed by flow cytometry. (L) Cells were incubated with or without \n10 mM 3-MA for the indicated times and counted using cell counter. (M–O) The cells were scratched with a \nT200 tip and then treated as in (L) for the indicated times. (M) Cells were stained using the CytoPainter Cell \nTracking Staining Kit and photographed. (N) The cell-free area was measured using ImageJ and change of cell-\nfree area was calculated. (O) The number of migrated cells was counted using ImageJ. Abbreviations: simulated \nmicrogravity (SM); propidium iodine (PI); 3-methyladenine (3-MA). Data are expressed as mean ± SD, with \npaired t-tests performed as indicated. *P < 0.05, **P < 0.01 versus control at each indicated time.\n\n7Scientific  RepoRtS  |         (2019) 9:12094  | https://doi.org/10.1038/s41598-019-48580-9\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nof differentiation (Fig. 7). These results suggest that Akt activity is crucial for maintaining the decidualization \npotential of eSCs. However, decrease in Akt phosphorylation was accompanied by neither phosphorylation of \nS6K1, 4EBP1 (mTORC1 activity; Fig. 3A), nor NDRG (mTORC2 activity; Fig. 3E). The composition of the mTOR \ncomplex was also not affected (Fig. 3E). These results suggested that another regulation pathway may induce the \ndecrease in Akt phosphorylation under SM conditions. Since the regulation of Akt activity by phosphatases, \nsuch as PP2A in endometrial cancer22 and FKBP51 in the decidualization of human eSCs23 has been reported in \nhuman eSCs, SM may activate Akt-specific phosphatases, which resulted in a decrease in Akt phosphorylation. \nWhether PP2A or FKBP51 is involved in SM-induced decrease in Akt activity warrants further investigation.\nRemodeling of the extracellular environment is a key event in decidualization. MMPs are responsible for \ncleaving the ECM components and process ECM-tethered growth factors for tissue remodeling24, which is crit-\nical for successful decidualization. Activin, a positive regulator of decidualization, promotes the expression of \nMMPs\n25, whereas TGF-β suppresses endometrial MMP activity26. MMP-9 also produces steroid response compo-\nnent 1-isoform C in a TNF-α dependent manner in the endometriotic mouse tissue27, suggesting that MMPs reg-\nulate endometrial physiology in diverse ways. We found that Akt inhibition induces slow migration by decreasing \nMMP expression in eSCs, as previously shown in melanoma\n28, breast cancer cells 29, vascular smooth muscle \ncells30, and human rheumatoid arthritis fibroblast-like synoviocytes 31. Whether Akt directly regulates MMP \nexpression needs to be further investigated in eSCs.\nβ-catenin is a transmembrane protein, which is associated with E-cadherin and involved in cell adhesion. \nβ-catenin is also a component of the Wnt signaling pathway 32. In the absence of a Wnt signal in normal cells, \nβ-catenin forms a complex, which includes glycogen synthase kinase 3β  (GSK-3β ). GSK-3β  phosphorylates \nβ-catenin, targeting it for ubiquitin-dependent degradation by the proteasome, thereby maintaining a low level \nof free cytoplasmic β -catenin32. In the present study, β -catenin phosphorylation in human eSCs decreased in \nan Akt-independent manner under SM conditions (Fig. 4G,H). A previous report shows that PTEN regulates \nnuclear localization and pSer675-β-catenin independent of the PI3K–AKT–GSK3b axis\n33. Thus, a novel regula-\ntory signaling of Wnt/ β-catenin independent of PI3K/Akt may exist, which warrants further investigation.\nThe expression of FOXO has been shown to be differentially regulated in human eSCs34. FOXO3a is expressed \nin undifferentiated eSCs, but not in decidualized eSCs. We found that exposure to SM significantly reduced \nFOXO3a expression (Fig. 5A), leading to a decrease in autophagic flux (Fig. 5J,K). FOXO3a is a requisite for sus-\ntaining autophagy under low nutrient conditions\n35. Basal autophagic flux is dependent on mTOR signaling, and \nthe induction or maintenance of autophagic flux was determined by FOXOs in muscle atrophy35. Because of the \nredundancy of FOXO family members in muscle cells, the deletion of a single member of the FOXO family does \nnot suppress autophagy\n35. However, SM exposure-induced FOXO3a deletion was sufficient to inhibit the expres-\nsion of autophagic genes and subsequent autophagic flux in undifferentiated eSCs. It is possible that FOXO3a is \nFigure 6. Both Akt and autophagic flux regulated cell migration under SM condition in human eSCs. (A) The \ncells were incubated with or without both 10 mM 3-MA and 1 μM Akti for the indicated times and counted \nusing a cell counter. (B–D) The cells were scratched with a T200 tip and treated as in (A) for the indicated times. \n(B) The cells were stained using the CytoPainter Cell Tracking Staining kit and photographed. (C) The cell-free \nareas were measured using ImageJ and changes in cell-free areas were calculated. (D) The number of migrated \ncells was counted using ImageJ. Abbreviations: simulated microgravity (SM); 3-methyladenine (3-MA). Data \nare expressed as mean ± SD, with paired t-tests performed as indicated. *P < 0.05, **P < 0.01 versus control at \neach indicated time.\n\n8Scientific  RepoRtS  |         (2019) 9:12094  | https://doi.org/10.1038/s41598-019-48580-9\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nthe main member of the FOXO family that maintains autophagy in undifferentiated eSCs, since FOXO3a is highly \nexpressed in those cells, while FOXO1 is expressed in the decidualized endometrium in vitro36 and in vivo34, and \nFOXO4 is not expressed in the normal endometrium 34. The role of autophagy in migration has been recently \ndemonstrated37. Inhibition of autophagy decreases the rate of cell motility by stabilizing focal adhesions, subse-\nquently resulting in the reduction of migration rate37. The association between autophagosome and focal adhe-\nsions facilitates the destabilization and turnover of cell-matrix contacts via focal adhesion proteins38. Decreased \nautophagic flux stabilizes cell-matrix contacts under SM conditions in eSCs and simultaneously, a low level of \nMMP-2 further inhibits cell matrix degradation.\nFigure 7. SM decreased decidualization of human eSCs. (A) Human eSCs were incubated either under 1 g or \nSM for 1 day, after which they were induced to differentiate in the presence of 0.5 mM 8-Br-cAMP under 1 g \nfor 1 day. Cells were lysed and subjected to a quantitative real time PCR (qRT-PCR) analysis. (B) The cells were \ntreated as in (A), induced to decidualization for 4 days, (C) stained using senescence associated β-galactosidase \nstaining kit, and photographed under microscope. Scale bar = 50 μm. (D) The cells were treated with 8-Br-\ncAMP under 1 g or SM for 1 day, lysed, and analyzed by qRT-PCR. (E) The cells were differentiated in the \npresence of 8-Br-cAMP for one day and shifted to either 1 g or SM for one day in the presence 8-Br-cAMP . The \ncells were then subjected to qRT-PCR. (F) The cells were differentiated as in (D) and shifted to either 1 g or SM \nfor one day in the absence of 8-Br-cAMP . (G) The cells were incubated under SM with or without 5 μg/ml SC-79 \nfor 1 day, after which they were induced to differentiate in the presence of 8-Br-cAMP under 1 g for 1 day. The \ncells were lysed and subjected to a qRT-PCR analysis. Abbreviations: simulated microgravity (SM); simulated \nmicrogravity for 24 h before the induction of differentiation (Pre-SM); prolactin (PRL); insulin-like growth \nfactor binding protein 1 (IGFBP1). Data are expressed as mean ± SD, with paired t-tests performed as indicated. \n°P < 0.05 versus undifferentiated control; *P < 0.05 versus differentiated control without Pre-SM; \n•P < 0.05 \nversus differentiated cells with Pre-SM.\n\n9Scientific  RepoRtS  |         (2019) 9:12094  | https://doi.org/10.1038/s41598-019-48580-9\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nIn the present study, we used specialized culture dishes, which are porous to air and completely sealed, in \norder to maintain the culture system during the rotation of human eSCs on the clinostat. Due to the lack of a \nsuitable culture system for the current clinostat, the current study has limitations, since we were unable to test the \neffect SM exposure on extracellular biomatrix invasion and the 3D culture of eSCs. As such, this warrants further \ninvestigation.\nMechanical unloading by exposure to SM altered cell growth as well as decidualization in eSCs. Our study \nprovides the first evidence that decidualization was restrained under SM conditions via a decrease in Akt activity \nand FOXO3a expression. The decrease in Akt activity and autophagic flux led to slow cell growth and migration, \nresulting in low efficiency of decidualization. Taken together, our findings suggest that the microgravity during \nspaceflight could lead to an unreceptive endometrium by suppressing decidualization potential.\nMethods\nAntibodies and other reagents. Antibodies were obtained as follows: anti-raptor and -rictor antibodies \nwere from Bethyl Laboratories (Montgomery, TX, USA); all other primary antibodies were from Cell Signaling \nTechnology (Danvers, MA, USA). All secondary antibodies were from Jackson ImmunoResearch Laboratories \nInc. (West Grove, PA, USA). All other reagents were from Sigma-Aldrich (St. Louis, MO, USA).\nIsolation and culture of human eSCs. Human eSCs were isolated from the human endometrium, which \nwas obtained by hysterectomy from 25 premenopausal women, aged 40–45 years. The participants underwent \nsurgery for non-endometrial abnormalities at Gil Hospital between August 2018 and January 2019. All pro-\ncedures were approved by Gachon University and the Institutional Review Board (IRB) (Permission number: \nGAIRB2018–301). All experiments were performed in accordance with the relevant guidelines and regulations. \nInformed consent was obtained from all participants. Isolation of eSCs was performed following a previously \ndescribed procedure\n17. Human eSCs were grown in Dulbecco’s modified Eagle’s medium (DMEM) contain-\ning 1 g/L glucose with 10% fetal bovine serum (FBS) at 37 °C and 5% CO 2 and detached from the plate using \n0.05% trypsin-EDTA (Welgene, Gyeongsangbuk-do, Korea). To induce in vitro decidualization, cells were plated, \ngrown to 100% confluence, treated with DMEM with 10% FBS containing 0.5 mM 8-Br-cAMP , and replen-\nished with fresh medium every other day. The cells were stained with senescence-associated β -galactosidase \n(senescence-associated β-galactosidase staining kit, Cell Signaling Technology).\nSM: the clinostat system. To induce SM on the ground, a clinostat system (3D clinostat, Shamhantech Inc., \nBucheon, Korea) was used in this study. Human eSCs were plated in a membrane cell culture dish (SPLPermea™; \nSPL Life Sciences Co., Gyeonggi-do, Korea). The cells were attached to the cell culture dish, which was filled with \nculture medium. The dish was fixed carefully to the rotating panel of the clinostat system, which was then placed \nin an incubator at 37 °C with a 5% CO\n2 atmosphere. The clinostat was continuously rotated at 5 rpm for 36 h. The \ncontrol cells (normal gravity) were plated on the same type of dish and incubated in the same incubator as the \ncells exposed to SM but did not undergo clinorotation.\nIn vitro scratch wound healing assay.  Human eSCs were incubated in DMEM containing 1.0 g/L glu-\ncose supplemented with 10% FBS. The medium was then replaced with DMEM (0.1% FBS), after which the cells \nwere incubated at 37 °C in an atmosphere of 5% CO\n2 for 18 h to minimize cell proliferation. An artificial wound \nwas created by disrupting the monolayer using a sterile plastic pipette tip (200 µL). A migration assay was then \nperformed in the presence or absence of SM at 6, 12, and 24 h. The cells were then stained using the CytoPainter \nFigure 8. The proposed model of the regulation of decidualization in human eSCs under SM. Exposure of \nhuman eSCs to SM decreased FOXO3a expression level and Akt activity, leading to the blockage of autophagic \nflux and MMP-2 expression, respectively. This reduced the growth and migration of human eSCs, resulting in \ndefective decidualization.\n\n10Scientific  RepoRtS  |         (2019) 9:12094  | https://doi.org/10.1038/s41598-019-48580-9\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\nCell Tracking Staining Kit (Abcam, Cambridge, MA, USA) following the manufacturer’s protocol. Images were \ncaptured using a laser Scanning Microscope 700 (Carl Zeiss, Oberkochen, Germany) equipped with a 5× objec-\ntive. Cell migration was measured as the percentage of the remaining wound area relative to the cell-free area of \nthe initial scratch. The number of migrated cells was calculated using an ImageJ cell counter. All experiments were \nperformed in at least triplicate.\nCell lysis, immunoprecipitation, and western blot analysis.  Human eSCs were washed once with \nice-cold phosphate buffered saline (PBS), scraped and then lysed with lysis buffer (Cell Signaling Technology). \nThe supernatant was collected after microcentrifugation at 13,000 g for 10 min, and then boiled in sodium dode-\ncyl sulfate sample buffer for 5 min. Immunoprecipitation was performed with anti-mTOR antibody, followed \nby incubation with protein G agarose for 1 h at 4 °C. For immunoprecipitation, lysis buffer containing 40 mM \n4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.4), 120 mM NaCl, 10 mM pyrophosphate, 50 mM NaF , \n10 mM β-glycerophosphate, 2 mM EDTA, 1X Sigma protease inhibitor cocktail, and 0.3% 3-[(3-cholamidopropyl)\ndimethylammonio]-1-propanesulfonate was used. Western blotting was performed as previously described\n36.\nQuantitative real-time (RT)-PCR.  Total RNA was extracted from human eSCs under either terrestrial \ngravity or SM. Quantitative RT-PCR was performed following a previously described protocol 36. Human glyc-\neraldehyde 3-phosphate dehydrogenase (GAPDH) was used to normalize gene expression. The following prim-\ners were used; PRL, Forward: GGAGCAAGCCCAACAGATGAA, Reverse: GGCTCATTCCAGGATCGCAAT; \nIGFBP1, Forward: TTGGGACGCCATCAGTACCTA, Reverse: TTGGCTAAACTCTCTACGACTCT; GAPDH, \nForward: GGAGCGAGATCCCTCCAAAAT, Reverse: GGCTGTTGTCATACTTCTCATGG.\nCell proliferation and viability. The number of trypan blue (Welgene, Gyeongsangbuk-do, Korea)-stained \ncells was counted to assess cell viability, according to the dye exclusion method39 using a cell counter (LUNA-II™ \nAutomated Cell Counter, Gyenggi-do, Korea). All counts were performed in duplicate with independent samples \nafter 12, 24, and 36 h of growth. To analyze cell viability, cells were collected by centrifugation at a concentration \nof 3 × 10\n5 cells/tube, incubated with either 7-AAD (50 μg/mL, Biolegend, San Diego, CA, USA) for 10 min at \nroom temperature, or PI (50 mg/L) and 1.5% of RNase A (7 mg/mL) for 30 min at 37 °C in the dark. The number \nof 7-AAD or PI-stained cells was counted using flow cytometry analysis (BD FACS Calibur; BD Biosciences, San \nJose, CA, USA).\nAnalysis of the cell cycle and apoptosis.  The cells were collected by centrifugation at a concentration \nof 3 × 105 cells/tube and washed twice with PBS after exposure to SM for 36 h. The cell pellets were suspended in \n1 mL ice-cold 70% ethanol at 4 °C for 1 h and washed with PBS once. The cells were then resuspended in 0.5 mL \nof PI (50 mg/L) and 1.5% of RNase A (7 mg/mL) for 30 min at 37 °C in the dark, and analyzed using flow cytom-\netry analysis (BD FACS Calibur; BD Biosciences). The cells were classified as late- or early-stage apoptotic cells \nby staining with annexin V-FITC and PI (FITC Annexin V apoptosis detection kit-1; BD Pharmingen, San Jose, \nCA, USA). Briefly, the cells were collected by centrifugation at a concentration of 3 × 10\n5 cells/tube, washed twice \nwith cold-PBS and once with 1 mL of binding buffer, and stained with 150 µL binding buffer containing 2.5 µL of \nannexin V-FITC and 0.1 µL of PI at room temperature for 15 min in the dark. The stained cells were subjected to \nflow cytometry analysis (BD FACS Calibur; BD Biosciences).\nReferences\n 1. Stein, T. P . Weight, muscle and bone loss during space flight: another perspective. Eur J Appl Physiol 113, 2171–2181 (2013).\n 2. Sonnenfeld, G. The immune system in space and microgravity. Medicine and science in sports and exercise 34, 2021–2027 (2002).\n 3. Norsk, P . Cardiovascular and fluid volume control in humans in space. Curr Pharm Biotechnol 6, 325–330 (2005).\n 4. Mandsager, K. T., Robertson, D. & Diedrich, A. The function of the autonomic nervous system during spaceflight. Clin Auton Res \n25, 141–151 (2015).\n 5. Macho, L. et al. Endocrine responses to space flights. J Gravit Physiol 8, P117–120 (2001).\n 6. Blaber, E. A. et al. Microgravity Reduces the Differentiation and Regenerative Potential of Embryonic Stem Cells. Stem Cells Dev 24, \n2605–2621 (2015).\n 7. Harada, M. et al. Mechanical stretch upregulates IGFBP-1 secretion from decidualized endometrial stromal cells. American journal \nof physiology. Endocrinology and metabolism 290, E268–272 (2006).\n 8. Harada, M. et al . Mechanical stretch stimulates interleukin-8 production in endometrial stromal cells: possible implications in \nendometrium-related events. J Clin Endocrinol Metab 90, 1144–1148 (2005).\n 9. Lyons, E. A. et al. Characterization of subendometrial myometrial contractions throughout the menstrual cycle in normal fertile \nwomen. Fertil Steril 55, 771–774 (1991).\n 10. Weimar, C. H., Macklon, N. S., Post Uiterweer, E. D., Brosens, J. J. & Gellersen, B. The motile and invasive capacity of human \nendometrial stromal cells: implications for normal and impaired reproductive function. Human reproduction update 19, 542–557 \n(2013).\n 11. Fang, D. et al. Phosphorylation of β-Catenin by AKT Promotes β-Catenin Transcriptional Activity. Journal of Biological Chemistry \n282, 11221–11229 (2007).\n 12. Chin, Y . R. & Toker, A. Function of Akt/PKB signaling to cell motility, invasion and the tumor stroma in cancer. Cellular signalling \n21, 470–476 (2009).\n 13. Saxton, R. A. & Sabatini, D. M. mTOR Signaling in Growth, Metabolism, and Disease. Cell 169, 361–371 (2017).\n 14. Long, J. et al. FOXO3a is essential for murine endometrial decidualization through cell apoptosis during early pregnancy. J Cell \nPhysiol 234, 4154–4166 (2019).\n 15. Zhang, X., Tang, N., Hadden, T. J. & Rishi, A. K. Akt, FoxO and regulation of apoptosis. Biochim Biophys Acta  1813, 1978–1986 \n(2011).\n 16. Brighton, P . J. et al. Clearance of senescent decidual cells by uterine natural killer cells in cycling human endometrium. Elife 6 (2017).\n 17. Y oon, M. S. et al. Phospholipase D1 as a key enzyme for decidualization in human endometrial stromal cells. Biol Reprod  76, \n250–258 (2007).\n 18. Ramathal, C. Y ., Bagchi, I. C., Taylor, R. N. & Bagchi, M. K. Endometrial decidualization: of mice and men. Semin Reprod Med 28, \n17–26 (2010).\n\n11Scientific  RepoRtS  |         (2019) 9:12094  | https://doi.org/10.1038/s41598-019-48580-9\nwww.nature.com/scientificreportswww.nature.com/scientificreports/\n 19. Bagchi, M. K., Mantena, S. R., Kannan, A. & Bagchi, I. C. Control of uterine cell proliferation and differentiation by C/EBPbeta: \nfunctional implications for establishment of early pregnancy. Cell Cycle 5, 922–925 (2006).\n 20. Enomoto, A. et al. Akt/PKB Regulates Actin Organization and Cell Motility via Girdin/APE. Developmental Cell 9, 389–402 (2005).\n 21. Fabi, F. et al . Regulation of the PI3K/Akt pathway during decidualization of endometrial stromal cells. PloS one 12, \ne0177387–e0177387 (2017).\n 22. Hsu, A. H. et al. Crosstalk between PKCalpha and PI3K/AKT Signaling Is Tumor Suppressive in the Endometrium. Cell Rep 24, \n655–669 (2018).\n 23. Wei, M. et al. FKBP51 regulates decidualization through Ser473 dephosphorylation of AKT. Reproduction 155, 283–295 (2018).\n 24. Lu, P ., Takai, K., Weaver, V . M. & Werb, Z. Extracellular matrix degradation and remodeling in development and disease. Cold Spring \nHarbor perspectives in biology 3, a005058, https://doi.org/10.1101/cshperspect.a005058 (2011).\n 25. Jones, R. L. et al. Activin A and inhibin A differentially regulate human uterine matrix metalloproteinases: potential interactions \nduring decidualization and trophoblast invasion. Endocrinology 147, 724–732 (2006).\n 26. Osteen, K. G., Igarashi, T. M. & Bruner-Tran, K. L. Progesterone action in the human endometrium: induction of a unique tissue \nenvironment which limits matrix metalloproteinase (MMP) expression. Frontiers in bioscience: a journal and virtual library 8, \nd78–86 (2003).\n 27. Han, S. J. et al. A new isoform of steroid receptor coactivator-1 is crucial for pathogenic progression of endometriosis. Nat Med 18, \n1102–1111 (2012).\n 28. Liang, X. et al. Rictor regulates the vasculogenic mimicry of melanoma via the AKT-MMP-2/9 pathway. Journal of Cellular and \nMolecular Medicine 21, 3579–3591 (2017).\n 29. Zhou, R. et al. Formononetin inhibits migration and invasion of MDA-MB-231 and 4T1 breast cancer cells by suppressing MMP-2 \nand MMP-9 through PI3K/AKT signaling pathways. Hormone and metabolic research =  Hormon- und Stoffwechselforschung = \nHormones et metabolisme 46, 753–760 (2014).\n 30. Wang, C. et al. Apelin induces vascular smooth muscle cells migration via a PI3K/Akt/FoxO3a/MMP-2 pathway. The international \njournal of biochemistry & cell biology 69, 173–182 (2015).\n 31. Yuan, H. et al. Knockdown of sphingosine kinase 1 inhibits the migration and invasion of human rheumatoid arthritis fibroblast-like \nsynoviocytes by down-regulating the PI3K/AKT activation and MMP-2/9 production in vitro . Molecular biology reports  41, \n5157–5165 (2014).\n 32. Aberle, H., Bauer, A., Stappert, J., Kispert, A. & Kemler, R. beta-catenin is a target for the ubiquitin-proteasome pathway. EMBO J \n16, 3797–3804 (1997).\n 33. Conde-Perez, A. et al. A caveolin-dependent and PI3K/AKT-independent role of PTEN in beta-catenin transcriptional activity. Nat \nCommun 6, 8093 (2015).\n 34. Kajihara, T. et al. Differential expression of FOXO1 and FOXO3a confers resistance to oxidative cell death upon endometrial \ndecidualization. Mol Endocrinol 20, 2444–2455 (2006).\n 35. Milan, G. et al. Regulation of autophagy and the ubiquitin–proteasome system by the FoxO transcriptional network during muscle \natrophy. Nature Communications 6, 6670 (2015).\n 36. Baek, M. O., Song, H. I., Han, J. S. & Y oon, M. S. Differential regulation of mTORC1 and mTORC2 is critical for 8-Br-cAMP-induced \ndecidualization. Exp Mol Med 50, 141 (2018).\n 37. Kenific, C. M. et al. NBR1 enables autophagy-dependent focal adhesion turnover. The Journal of Cell Biology 212, 577–590 (2016).\n 38. Kenific, C. M., Wittmann, T. & Debnath, J. Autophagy in adhesion and migration. Journal of cell science 129, 3685–3693 (2016).\n 39. Yip, D. K. & Auersperg, N. The dye-exclusion test for cell viability: persistence of differential staining following fixation. In Vitro 7, \n323–329 (1972).\nAcknowledgements\nThis work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean \ngovernment (Ministry of Science and ICT;2018R1A2B6004513) and the Korea Health Technology R&D Project \nthrough the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare \n(HI17C0426).\nAuthor contributions\nH.-J.C. and M.-S.Y . originated the idea; H.-J.C., M.-O.B. and S.A.K. conducted the experiments; S.J.C. provides \nhuman samples; K.H.S. and S.H.L. provided the clinostat; H.-J.C. and M.-S.Y . analyzed the results; M.-S.Y . wrote \nthe manuscript; and H.-J.C. and M.-S.Y . reviewed and edited the manuscript. All authors read and approved the \nfinal manuscript.\nAdditional Information\nSupplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-48580-9.\nCompeting Interests: The authors declare no competing interests.\nPublisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and \ninstitutional affiliations.\nOpen Access This article is licensed under a Creative Commons Attribution 4.0 International \nLicense, which permits use, sharing, adaptation, distribution and reproduction in any medium or \nformat, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-\native Commons license, and indicate if changes were made. The images or other third party material in this \narticle are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the \nmaterial. If material is not included in the article’s Creative Commons license and your intended use is not per-\nmitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the \ncopyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.\n \n© The Author(s) 2019","source_license":"CC0","license_restricted":false}