LY6D-induced macropinocytosis as a survival mechanism of senescent cells

Abstract

Although senescent cells display various morphological changes including vacuole formation, it is still unclear how these processes are regulated. We have recently identified the gene, lymphocyte antigen 6 complex, locus D (LY6D), to be upregulated specifically in senescent cells. LY6D is a glycosylphosphatidylinositol-anchored cell-surface protein whose function remains unknown. Here, we analyzed the functional relationship between LY6D and the senescence processes. We found that overexpression of LY6D induced vacuole formation and knockdown of LY6D suppressed the senescence-associated vacuole formation. The LY6D-induced vacuoles were derived from macropinocytosis, a distinct form of endocytosis. Furthermore, Src family kinases and Ras were found to be recruited to membrane lipid rafts in an LY6D-dependent manner, and inhibition of their activity impaired the LY6D-induced macropinocytosis. Finally, reduction of senescent-cell survival induced by glutamine deprivation was recovered by albumin supplementation to the culture media in an LY6D-dependent manner. Because macropinocytosis acts as an amino acid supply route, these results suggest that LY6D-mediated macropinocytosis contributes to senescent-cell survival through the incorporation of extracellular nutrients. Although senescent cells display various morphological changes including vacuole formation, it is still unclear how these processes are regulated. We have recently identified the gene, lymphocyte antigen 6 complex, locus D (LY6D), to be upregulated specifically in senescent cells. LY6D is a glycosylphosphatidylinositol-anchored cell-surface protein whose function remains unknown. Here, we analyzed the functional relationship between LY6D and the senescence processes. We found that overexpression of LY6D induced vacuole formation and knockdown of LY6D suppressed the senescence-associated vacuole formation. The LY6D-induced vacuoles were derived from macropinocytosis, a distinct form of endocytosis. Furthermore, Src family kinases and Ras were found to be recruited to membrane lipid rafts in an LY6D-dependent manner, and inhibition of their activity impaired the LY6D-induced macropinocytosis. Finally, reduction of senescent-cell survival induced by glutamine deprivation was recovered by albumin supplementation to the culture media in an LY6D-dependent manner. Because macropinocytosis acts as an amino acid supply route, these results suggest that LY6D-mediated macropinocytosis contributes to senescent-cell survival through the incorporation of extracellular nutrients. Cellular senescence is defined as an irreversible cell proliferation arrest induced by various stresses, such as telomere erosion, activated oncogenes, oxidative stress, and DNA damage (1Campisi J. Aging, cellular senescence, and cancer.Annu. Rev. Physiol. 2013; 75: 685-705Crossref PubMed Scopus (1315) Google Scholar, 2Salama R. Sadaie M. Hoare M. Narita M. Cellular senescence and its effector programs.Genes Dev. 2014; 28: 99-114Crossref PubMed Scopus (433) Google Scholar). It has been first described in primary human fibroblasts and now considered to play a critical role in tumor suppression and aging-related diseases including cancer. Senescent cells are known to display a variety of morphological changes including a formation of cytoplasmic vacuoles. However, despite the early discovery of senescence-associated vacuole formation in 1970s (3Comings D.E. Okada T.A. Electron microscopy of human fibroblasts in tissue culture during logarithmic and confluent stages of growth.Exp. Cell Res. 1970; 61: 295-301Crossref PubMed Scopus (57) Google Scholar, 4Lipetz J. Cristofalo V.J. Ultrastructural changes accompanying the aging of human diploid cells in culture.J. Ultrastruct. Res. 1972; 39: 43-56Crossref PubMed Scopus (130) Google Scholar), it has remained unclear how this process is regulated during senescence and what the physiological significance is. Lymphocyte antigen 6 complex, locus D (LY6D) is a membrane-bound protein attached to the cell surface via a C-terminal glycosylphosphatidylinositol (GPI) anchor (5Lee P.Y. Wang J.X. Parisini E. Dascher C.C. Nigrovic P.A. Ly6 family proteins in neutrophil biology.J. Leukoc. Biol. 2013; 94: 585-594Crossref PubMed Scopus (128) Google Scholar). Although LY6D is often used as a surface marker for leukocyte subset identification because of its lineage-specific expression, the physiological function of LY6D is poorly understood. We have recently identified LY6D to be upregulated specifically in senescent cells by comparing the transcriptome between senescent and apoptotic cells and shown that the upregulation of LY6D is dependent on p53, a crucial transcription factor for the initiation and maintenance of senescence (6Nagano T. Nakano M. Nakashima A. Onishi K. Yamao S. Enari M. Kikkawa U. Kamada S. Identification of cellular senescence-specific genes by comparative transcriptomics.Sci. Rep. 2016; 6: 31758Crossref PubMed Scopus (30) Google Scholar, 7Nagano T. Nakashima A. Onishi K. Kawai K. Awai Y. Kinugasa M. Iwasaki T. Kikkawa U. Kamada S. Proline dehydrogenase promotes senescence through the generation of reactive oxygen species.J. Cell Sci. 2017; 130: 1413-1420Crossref PubMed Scopus (19) Google Scholar, 8Nagano T. Yamao S. Terachi A. Yarimizu H. Itoh H. Katasho R. Kawai K. Nakashima A. Iwasaki T. Kikkawa U. Kamada S. D-amino acid oxidase promotes cellular senescence via the production of reactive oxygen species.Life Sci. Alliance. 2019; 2e201800045Crossref PubMed Scopus (4) Google Scholar). However, it is still unknown whether LY6D functionally contributes to the senescence process because our previous study has shown that ectopic expression of LY6D in osteosarcoma U2OS cells had little effect on senescence induction as determined by colony-forming assay and by senescence-associated β-galactosidase (SA-β-Gal) staining, an established marker of senescence. GPI-anchored proteins are known to show dynamic behaviors in terms of their diffusion, organization, and interactions membrane and the a of GPI-anchored proteins is in of the membrane to the extracellular to effector in the Sci. 75: PubMed Scopus Google Scholar, K. rafts and Rev. Cell Biol. PubMed Scopus Google Scholar). lipid rafts as for dynamic between GPI-anchored and The functional of of these proteins in the lipid rafts has been in for in the of Src family kinases S. S. GPI-anchored protein and the cell Res. 2016; PubMed Scopus Google Scholar). GPI-anchored such as and to the and of H. GPI-anchored cell-surface to protein PubMed Scopus Google Scholar, R. M. A. GPI-anchored and for and study Cell Biol. PubMed Scopus Google Scholar, Y. K. of proteins on Src family and Cell Biol. PubMed Scopus Google Scholar). However, in to these GPI-anchored the functional of LY6D in the of has been as is a and process by of extracellular and are cells through vacuoles of formation and J. 2017; PubMed Scopus Google Scholar). macropinocytosis is regulated by interactions by such as and of these the cell in promotes membrane and formation. has been in and because of its role in the through the of extracellular it has been recently that the of from extracellular cells to in the of and amino is of formation and J. 2017; PubMed Scopus Google Scholar, S. E. M. of protein is an amino acid supply in 2013; PubMed Scopus Google Scholar). the in activity between and senescent cells R. Sadaie M. Hoare M. Narita M. Cellular senescence and its effector programs.Genes Dev. 2014; 28: 99-114Crossref PubMed Scopus (433) Google Scholar), it has been whether macropinocytosis contributes to survival of senescent cells to the we the relationship between LY6D and the senescence We have found that LY6D is for the senescence-associated vacuole formation, through the induction of macropinocytosis. and Ras were in the membrane lipid rafts senescence in an LY6D-dependent manner. Furthermore, LY6D-induced macropinocytosis survival of senescent cells by the of extracellular the function of LY6D in the senescence an expression LY6D was and human osteosarcoma U2OS cells LY6D amino was as we the cells cytoplasmic vacuole formation overexpression of LY6D The vacuole formation is known as a of senescence (3Comings D.E. Okada T.A. Electron microscopy of human fibroblasts in tissue culture during logarithmic and confluent stages of growth.Exp. Cell Res. 1970; 61: 295-301Crossref PubMed Scopus (57) Google Scholar, 4Lipetz J. Cristofalo V.J. Ultrastructural changes accompanying the aging of human diploid cells in culture.J. Ultrastruct. Res. 1972; 39: 43-56Crossref PubMed Scopus (130) Google Scholar). of U2OS cells and that DNA induced the vacuole formation in the an established marker of senescence as by senescent cells were identified as cells and Furthermore, the vacuole formation senescence was in human diploid fibroblasts cells and in the LY6D was to be upregulated during senescence (6Nagano T. Nakano M. Nakashima A. Onishi K. Yamao S. Enari M. Kikkawa U. Kamada S. Identification of cellular senescence-specific genes by comparative transcriptomics.Sci. Rep. 2016; 6: 31758Crossref PubMed Scopus (30) Google Scholar). results the that LY6D is in the senescence-associated vacuole formation of tumor and cells. we LY6D by in U2OS cells of LY6D suppressed the vacuole formation during senescence and it had effect on activity and cell proliferation and and results were in cells and D and by the LY6D the upregulation of LY6D in cells in results suggest that LY6D is for the of senescence-associated vacuoles for the induction of senescence vacuole formation during senescence, its to U2OS cells for LY6D were to The LY6D protein to the were and are the of and U2OS cells for LY6D and for were to of cells and senescence The of cells and cells is cells for LY6D and for were to of cells and the LY6D protein to the were and are the of and U2OS cells and LY6D were to the and of cells of U2OS cells were by The to from the to the of the were to was used as a that the The results of are shown in U2OS cells were for and to of cells. and U2OS cells and were to the and of cells are significance is shown the lymphocyte antigen 6 complex, locus senescence-associated we an LY6D a of amino to the shown in was the cell the of LY6D (5Lee P.Y. Wang J.X. Parisini E. Dascher C.C. Nigrovic P.A. Ly6 family proteins in neutrophil biology.J. Leukoc. Biol. 2013; 94: 585-594Crossref PubMed Scopus (128) Google Scholar), the of the the cell-surface of Furthermore, overexpression of LY6D to the vacuole formation and to that the LY6D the vacuole formation. Because GPI-anchored proteins are known to be to cell membrane lipid S. S. GPI-anchored protein and the cell Res. 2016; PubMed Scopus Google Scholar, of formation and J. 2017; PubMed Scopus Google Scholar), we whether LY6D is to the this U2OS cell were by K. K. K. S. Iwasaki T. Y. Y. K. Y. identification and of an protein that is Biol. PubMed Scopus Google Scholar). shown in and LY6D was in the of cells as determined by as a Furthermore, LY6D was in the of cells that LY6D was upregulated in to DNA damage and to the the functional of LY6D on vacuole formation, the cells LY6D were a that the lipid rafts by of membrane K. rafts and Rev. Cell Biol. PubMed Scopus Google Scholar), and vacuole formation was The impaired the LY6D-induced vacuole formation in a reduction in the protein of LY6D and in the and the relationship between the LY6D and vacuole formation, we of and the C-terminal including the was the of and and were on the of their The of lipid rafts in the of Cell Sci. PubMed Google Scholar). The membrane of and was by assay overexpression of and to vacuole formation results suggest that the cell-surface of in the contributes to vacuole formation. We to the of LY6D-induced vacuoles. the between senescence and a process that of the of cytoplasmic proteins and by cytoplasmic vacuoles and their through R. Sadaie M. Hoare M. Narita M. Cellular senescence and its effector programs.Genes Dev. 2014; 28: 99-114Crossref PubMed Scopus (433) Google Scholar), we first that LY6D-induced vacuoles are derived from this the formation of in the cells was a Although the was in of the LY6D-induced vacuoles was an of used to to the LY6D-induced vacuole formation we the effect of knockdown of a gene, on the vacuole formation. The expression was suppressed by the of Furthermore, was as an knockdown of formation and the suppression of by knockdown of to the LY6D-induced vacuole formation we that the LY6D-induced vacuoles were derived from It has been that Ras cytoplasmic vacuole formation of membrane and in fibroblasts by PubMed Scopus Google and that the vacuoles are derived from macropinocytosis A. in cells through of Res. 6: PubMed Scopus Google Scholar). to whether LY6D the we the effect of acid a Ras on the LY6D-induced vacuole formation. the vacuole formation induced by LY6D overexpression the that LY6D-induced vacuoles are derived from macropinocytosis. this we the cells a marker S. E. M. of protein is an amino acid supply in 2013; PubMed Scopus Google Scholar). We that the LY6D-induced vacuoles were for the of and LY6D were that LY6D to be the vacuoles the senescence-associated vacuoles induced by the was by LY6D knockdown we the effect of a of used to macropinocytosis of of J. PubMed Scopus Google Scholar), on the LY6D-induced vacuole formation. The the vacuole formation induced by LY6D vacuole formation was by in U2OS and cells and Finally, a of for macropinocytosis Cell Biol. PubMed Scopus Google Scholar), the LY6D-induced that the LY6D-induced vacuole formation was by macropinocytosis. We to the the LY6D-induced vacuole formation. Because GPI-anchored proteins are known to form the S. S. GPI-anchored protein and the cell Res. 2016; PubMed Scopus Google Scholar, A. the lipid of and its J. PubMed Scopus Google Scholar), we whether the LY6D proteins in the cells by this we LY6D an between amino acid and because the of LY6D is of the protein and be by The and were in U2OS and was a of the LY6D the was by that LY6D in the cells LY6D has a that is by and from the is in the the of LY6D are whether the formation of LY6D to the vacuole formation we LY6D in the and were by and and because the of the in a of the was shown to the of to its J. J. J. of the Cell PubMed Scopus Google Scholar, A. the Ly6 of are crucial for the of Biol. PubMed Scopus Google Scholar). that formation of LY6D was in the and that these and play a role in the LY6D Furthermore, vacuole formation was induced by overexpression of the overexpression of the in a in the of cells the the of LY6D and results suggest that the of LY6D is for the induction of vacuole formation. Because it has been shown that the of GPI-anchored proteins as for proteins such as S. S. GPI-anchored protein and the cell Res. 2016; PubMed Scopus Google Scholar, H. GPI-anchored cell-surface to protein PubMed Scopus Google Scholar, R. M. A. GPI-anchored and for and study Cell Biol. PubMed Scopus Google Scholar, Y. K. of proteins on Src family and Cell Biol. PubMed Scopus Google Scholar), we whether such as and are in the The that Ras was in the LY6D was Furthermore, the from to were an in the to that in the lipid rafts LY6D expression because have in the of to in the was by LY6D overexpression as was a of the of and a the family and were found to be in the of cells. the induced the of and Ras to the of were by of LY6D and results suggest that a of proteins including and Ras is in the lipid during senescence in an LY6D-dependent manner. to the functional between the of and macropinocytosis, we the of various Ras on macropinocytosis M. cytoplasmic protein PubMed Scopus Google Scholar, of cell proliferation by a protein for Biol. Scholar). of form of Ras the Ras induced vacuole formation and the vacuole formation was by the of that macropinocytosis induced by Ras is dependent on Ras activity as A. in cells through of Res. 6: PubMed Scopus Google Scholar). the functional between LY6D and U2OS cells were LY6D and of the Ras and the vacuole formation was a and the LY6D-induced macropinocytosis, impaired that LY6D and Ras function in the and Ras acts of LY6D to macropinocytosis. a suppressed the LY6D-induced macropinocytosis the results that the to macropinocytosis. We to the that acts of to macropinocytosis. Ras such as and A. A. M. Ras to cell PubMed Scopus Google Scholar, distinct of Ras Ras effector that Ras and and cell Biol. PubMed Scopus Google Scholar). We found that the a the LY6D-induced macropinocytosis that the is for the induction of macropinocytosis. to is for the LY6D-induced vacuole formation, we Ras and and that of the and A. A. M. Ras to cell PubMed Scopus Google Scholar), and their effect on macropinocytosis. a overexpression of the the and induced vacuole formation the that the induced macropinocytosis. the previous that the and that overexpression of the activated the as by an in the of a effector of inhibition by and suppressed LY6D-induced and macropinocytosis results suggest that the macropinocytosis through the of the We to the physiological significance of senescence-associated induction of macropinocytosis. has been recently to function as an amino acid supply in cells of formation and J. 2017; PubMed Scopus Google Scholar, S. E. M. of protein is an amino acid supply in 2013; PubMed Scopus Google Scholar). have shown that a glutamine in survival of cells is recovered by extracellular supplementation albumin in a manner, to the that macropinocytosis contributes to cell survival through the incorporation of extracellular S. E. M. of protein is an amino acid supply in 2013; PubMed Scopus Google Scholar). the in the between and senescent cells R. Sadaie M. Hoare M. Narita M. Cellular senescence and its effector programs.Genes Dev. 2014; 28: 99-114Crossref PubMed Scopus (433) Google Scholar), it is that incorporation of extracellular survival of senescent cells as is the in cells. We whether LY6D-induced macropinocytosis survival of senescent cells Cell was by of culture in a in a We found that cell was in the in as S. E. M. of protein is an amino acid supply in 2013; PubMed Scopus Google Scholar). the in cell survival was recovered by the of the supplementation and LY6D overexpression 6 in by LY6D in show the of supplementation on cell the of were in in of cells to supplementation are results the that LY6D-induced macropinocytosis contributes to cell survival through the of the extracellular this in cells the senescence we whether the supplementation cell in senescent cells U2OS cells were for and in the for cells were to the of senescent cells by glutamine deprivation in was by the supplementation 6 in and in Because the by of senescent cells the glutamine these results suggest that senescence-associated macropinocytosis senescent cell survival through the incorporation of extracellular this a cell was by the in cell is by the activity of the results from staining, the assay that the in survival of senescent cells was recovered by the supplementation 6 in and in Furthermore, to whether the effect of supplementation on in senescent cell survival is dependent on the of was between and cells knockdown of LY6D impaired the survival by the in and in Finally, the was cells Senescent cells were to the cells in was by the supplementation as is the in U2OS cells in the of senescent cell was dependent on LY6D in and in results that senescence-associated macropinocytosis induced by LY6D contributes to survival of senescent cells in a Although senescent cells various changes such as and cell and vacuole formation, the are unclear (1Campisi J. Aging, cellular senescence, and cancer.Annu. Rev. Physiol. 2013; 75: 685-705Crossref PubMed Scopus (1315) Google R. Sadaie M. Hoare M. Narita M. Cellular senescence and its effector programs.Genes Dev. 2014; 28: 99-114Crossref PubMed Scopus (433) Google Scholar). We have found that LY6D is upregulated in and senescent the physiological of LY6D in the senescence (6Nagano T. Nakano M. Nakashima A. Onishi K. Yamao S. Enari M. Kikkawa U. Kamada S. Identification of cellular senescence-specific genes by comparative transcriptomics.Sci. Rep. 2016; 6: 31758Crossref PubMed Scopus (30) Google Scholar). LY6D is an extracellular GPI-anchored protein attached to the surface of the whose physiological function still remains (5Lee P.Y. Wang J.X. Parisini E. Dascher C.C. Nigrovic P.A. Ly6 family proteins in neutrophil biology.J. Leukoc. Biol. 2013; 94: 585-594Crossref PubMed Scopus (128) Google Scholar). the we that LY6D a role in the senescence-associated vacuole formation of tumor and cells. Furthermore, the LY6D-induced vacuole formation through the induction of macropinocytosis, a distinct form of endocytosis. macropinocytosis is of and and by the formation of cytoplasmic of formation and J. 2017; PubMed Scopus Google Scholar). the is considered to be by of to in such as and to However, we that the LY6D in the vacuole formation of factor results suggest that macropinocytosis be by the factor in this by the upregulation and of GPI-anchored Furthermore, we by that LY6D was in the lipid in to DNA The lipid rafts are for such as and and in the Sci. 75: PubMed Scopus Google Scholar, K. rafts and Rev. Cell Biol. PubMed Scopus Google Scholar, T. R. the function of J. Cell Biol. PubMed Scopus (130) Google Scholar). that GPI-anchored proteins are in the rafts to the S. S. GPI-anchored protein and the cell Res. 2016; PubMed Scopus Google Scholar, of formation and J. 2017; PubMed Scopus Google Scholar, A. A. lipid and in 2017; PubMed Scopus Google Scholar). a GPI-anchored membrane in the the between and its A. the lipid of and its J. PubMed Scopus Google Scholar). that and LY6D the a in (5Lee P.Y. Wang J.X. Parisini E. Dascher C.C. Nigrovic P.A. Ly6 family proteins in neutrophil biology.J. Leukoc. Biol. 2013; 94: 585-594Crossref PubMed Scopus (128) Google Scholar), it is that the LY6D its to functional and to macropinocytosis. this is the and Ras are the for the macropinocytosis from LY6D to the because and Ras were to be in the rafts senescence in an LY6D-dependent manner. we found that the inhibition of Ras impaired LY6D-induced macropinocytosis, and the inhibition suppressed LY6D-induced macropinocytosis that between LY6D and that LY6D macropinocytosis through the of our have been to be activated by the of GPI-anchored proteins in the H. GPI-anchored cell-surface to protein PubMed Scopus Google Scholar, R. M. A. GPI-anchored and for and study Cell Biol. PubMed Scopus Google Scholar, Y. K. of proteins on Src family and Cell Biol. PubMed Scopus Google Scholar), and the and Ras are known to be of the of membrane and in fibroblasts by PubMed Scopus Google Scholar, A. in cells through of Res. 6: PubMed Scopus Google Scholar, M. A. S. M. Src and macropinocytosis the surface of PubMed Scopus Google Scholar). Although are of the role of GPI-anchored proteins in the of are known to Ras through the M. J. R. A. S. J. J. T. of the and proteins is in of the Ras by PubMed Scopus Google Scholar). the Ras including and A. A. M. Ras to cell PubMed Scopus Google Scholar, distinct of Ras Ras effector that Ras and and cell Biol. PubMed Scopus Google Scholar). these we identified as the effector for the induction of macropinocytosis, by Ras that specifically of the Furthermore, the inhibition of suppressed LY6D-induced and macropinocytosis. these has been to to macropinocytosis and of Cell Sci. 2016; PubMed Scopus Google Scholar). is activated by Ras through its and the membrane lipid effector such as and a of proteins that have in in previous by suggest that the LY6D-induced Ras to the and to the induction of macropinocytosis. the of senescent cells are known to have a R. Sadaie M. Hoare M. Narita M. Cellular senescence and its effector programs.Genes Dev. 2014; 28: 99-114Crossref PubMed Scopus (433) Google Scholar). senescent cells display an proteins cell and an of study has that cells and extracellular proteins through macropinocytosis to their S. E. M. of protein is an amino acid supply in 2013; PubMed Scopus Google Scholar). the between and senescent cells in terms of the it is that LY6D-induced macropinocytosis contributes to the of senescent cells. of this we that the extracellular supplementation of the glutamine in senescent cell survival in an LY6D-dependent manner. have shown the between LY6D expression and in such as and and S. R. Y. lymphocyte 6 family and and 2016; PubMed Scopus Google Scholar). the on cell it is that the LY6D-mediated macropinocytosis cell survival in senescent in cells. Because the functional of LY6D to tumor maintenance has to be our study to a of the of cells. whether LY6D promotes the senescent cell survival in and whether LY6D macropinocytosis in cells are the for of their our results show that LY6D is for the senescence-associated vacuole formation through the induction of macropinocytosis, on the LY6D in the survival of senescent cells. U2OS human osteosarcoma and human diploid Cell are for to the of PubMed Google cells were in The cells were to DNA senescence U2OS and cells were and for and in the the for to senescent (6Nagano T. Nakano M. Nakashima A. Onishi K. Yamao S. Enari M. Kikkawa U. Kamada S. Identification of cellular senescence-specific genes by comparative transcriptomics.Sci. Rep. 2016; 6: 31758Crossref PubMed Scopus (30) Google Scholar, 7Nagano T. Nakashima A. Onishi K. Kawai K. Awai Y. Kinugasa M. Iwasaki T. Kikkawa U. Kamada S. Proline dehydrogenase promotes senescence through the generation of reactive oxygen species.J. Cell Sci. 2017; 130: 1413-1420Crossref PubMed Scopus (19) Google Scholar, 8Nagano T. Yamao S. Terachi A. Yarimizu H. Itoh H. Katasho R. Kawai K. Nakashima A. Iwasaki T. Kikkawa U. Kamada S. D-amino acid oxidase promotes cellular senescence via the production of reactive oxygen species.Life Sci. Alliance. 2019; 2e201800045Crossref PubMed Scopus (4) Google Scholar, M. Nakashima A. T. S. Kikkawa U. Kamada S. amino senescence through of upregulation of 2013; PubMed Scopus Google Scholar). expression was to the was used to was used to macropinocytosis of of J. PubMed Scopus Google Scholar). staining, the cells were for was to the and 6 was used to study macropinocytosis. was used to and from were used to D was media was and of and D were in and were in the of an expression for human the LY6D was a of and a a from U2OS cells as a The were and and The of LY6D was the and a and the to the of LY6D an between amino acid and DNA to the of LY6D the and the of LY6D were in this to the by the to and the of of DNA to the of and the of human were to the by the to and The human was from the generation of and and were and a for and a and a for as the to and the expression and that human and the were a of and a for and as the from K. The were and and of the in the the of and and were and a for a and a for and a and a for as the to and and were from and were from Cell and were from and were from and were from was from was from was from was from The cells were in the and the were by and membrane protein was primary as and the of the senescence β-galactosidase was used to the the cells were for and for the cells were Senescent cells were identified as cells and cells in were to the of cells. incorporation U2OS cells were for and incorporation was the to the The cells were for LY6D and its were from was from were and to the the cells were and in and primary in by the for cell the cells were cells were in and the were and for The were of The were in an of and of was the The were for in an the of were from the to the of the was as was as K. K. K. S. Iwasaki T. Y. Y. K. Y. identification and of an protein that is Biol. PubMed Scopus Google Scholar). cells were in The were of for The proteins were by of the The cell and were by and the Cell was by by a cell proliferation staining, cells were in a for to and U2OS cells were cells in a and for by the of to the and for The of the by was by The was used to for are in the The that have of the of this We K. for the We and for their S. Kamada and the T. T. K. Y. A. S. S. R. K. and S. Kamada the T. T. K. A. K. and S. Kamada analyzed the T. T. K. A. K. U. and S. Kamada T. and S. Kamada the the results and the of the was by for the of and The and a from a

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