The LY6D protein levels relative to the -tubulin levels were quantified using NIH ImageJ software and are indicated in the of each lane. 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 tradition media in an LY6D-dependent manner. Because macropinocytosis functions as an amino acid supply route, these results suggest that LY6D-mediated macropinocytosis contributes to senescent-cell survival through the incorporation of extracellular nutrients. and and gene was previously reported to be upregulated during senescence (6). These results raised the possibility that LY6D is definitely involved in the senescence-associated vacuole formation of both tumor and normal PD-1-IN-22 cells. To confirm this, we silenced LY6D by using siRNA in U2OS cells (Fig.?2and Fig.?S1A), whereas it had no effect on both SA–Gal activity and cell proliferation capacity (Fig.?2and Fig.?S1, and and Fig.?S1, and inhibited the etoposide-induced upregulation of LY6D in Hs68 cells (compare lanes 3 with 4 in Fig.?2indicate examples of cytoplasmic vacuoles. and indicate examples of cytoplasmic vacuoles. and indicate examples of cytoplasmic vacuoles. were subjected to immunoblot analysis. The LY6D protein levels relative to the -tubulin levels were quantified using NIH ImageJ software and are indicated in the of each lane. and and treated with 2-M etoposide for 7 days were subjected to quantification PD-1-IN-22 of vacuole-forming cells (and treated with 0.5-M etoposide for 7 days PPARGC1 were subjected to immunoblot analysis (of each lane. and and were subjected to immunoblot analysis ( 0.05). LY6D, lymphocyte antigen 6 complex, locus D; SA–Gal, senescence-associated -galactosidase. Localization of LY6D in the membrane lipid raft is required for vacuole formation Next, we generated an LY6D mutant (1-20 LY6D) harboring a deletion of N-terminal 20 amino acids corresponding to the transmission sequence (Fig.?1and Fig.?S1and Fig.?S6and Fig.?S1inhibited GFP-LC3 puncta formation less than normal growth conditions and serum starvation, indicating the successful suppression of autophagy by knockdown (Fig.?3failed to inhibit the LY6D-induced vacuole formation (Fig.?3indicate examples of cytoplasmic vacuoles. Bars, 20 m. and with pcDNA3-HA-LY6D were subjected to immunoblot analysis. and with pBABEpuro GFP-LC3 as an autophagy marker, cultured in either the growth medium (10% FBS) or serum starvation medium (0.5% FBS) for 18 h, and subjected to quantification of LC3-dotCpositive cells. Representative microscopic images (were subjected to quantification of PD-1-IN-22 vacuole-forming cells. PD-1-IN-22 Representative microscopic images ( 0.05). LY6D, lymphocyte antigen 6 complex, locus D; 3-MA, 3-methyladenine; FBS, fetal bovine serum. It has been reported that oncogenic Ras stimulates cytoplasmic vacuole formation (19) and that the Ras-induced vacuoles are derived from macropinocytosis (20). Consequently, to determine whether LY6D activates the Ras-mediated macropinocytic pathway, we tested the effect of farnesyl thiosalicylic acid (FTS), a Ras inhibitor, within the LY6D-induced vacuole formation. FTS efficiently inhibited the vacuole formation induced by LY6D overexpression (Fig.?4and indicate examples of cytoplasmic vacuoles. siRNA and treated with 2-M etoposide. After 7-day time treatment, the cells were incubated with dextran-Alexa Fluor 488 (10,000 MW) for 16 h and observed under fluorescence microscope. Representative microscopic images (show the colocalization of cytoplasmic vacuoles and fluorescent dextran. 0.05, ??of each lane. The results of different batch experiments are demonstrated in Fig.?S6and Fig.?S6of each lane. Data are mean? S.D. ( 0.05). LY6D, lymphocyte antigen 6 complex, locus D. We next set out to elucidate the signaling pathway that functions downstream of LY6D-SFK-Ras to induce macropinocytosis. Ras can activate several different downstream pathways such as MAP kinase, Ral, and PI3K pathways (28, 29). We found that the treatment with U0126, a MAPKK inhibitor, did not suppress the LY6D-induced macropinocytosis (Fig.?S5have shown that a glutamine deprivationCinduced decrease in survival of Ras-transformed cells is definitely recovered by extracellular supplementation with bovine serum albumin (BSA) inside a macropinocytosis-dependent manner, leading to the conclusion that Ras-induced macropinocytosis contributes to malignancy cell survival through the.