These results indicate that HT-1080, MDA-MB-231, and HepG2 cells possess the ability to attach to these polymer substrates via integrin-independent attachment without adsorbed proteins. Open in a separate window Fig 6 Integrin-independent attachment.(A) HT-1080, (B) MDA-MB-231, and HepG2 were incubated with the substrates in either serum-free (FBS(-)) or serum-containing (FBS(+)) media for 1 h. through the regulation of protein adsorption. In the present study, we NMS-1286937 investigated protein adsorption, cell attachment profiles, and attachment mechanisms on PMEA analogous polymer substrates. Additionally, we demonstrated the possibility of attachment-based cell enrichment on PMEA analogous polymer substrates. HT-1080 and MDA-MB-231 cells started to attach to poly(butyl acrylate) (PBA) and poly(tetrahydrofurfuryl acrylate) (PTHFA), on which proteins could adsorb well, within 1 h. HepG2 cells started to attach after 1 h. HT-1080, MDA-MB-231, and HepG2 cells started to attach within 30 min to PMEA, poly(2-(2-methoxyethoxy) ethyl acrylate-< 0.005 < 0.05 < 0.01 < 0.01, ***: < 0.005 < 0.05, **: < 0.01, ***: < 0.005 < 0.05, ***: < 0.005 < 0.05, ***: < 0.005 and were obviously expressed in HT-1080 and MDA-MB-231 cells, whereas these genes were expressed at lower levels in HepG2 cells. These results indicate that the integrin-dependent attachments of HT-1080 and MDA-MB-231 cells were stronger than those of HepG2 cells because of the difference in integrin expression. In addition to characterizing the integrin-dependent attachment of these cells, we also compared the characteristics of integrin-independent attachment. We performed a cell attachment assay in a serum-free medium DUSP2 after 1 h (Fig 6). The HT-1080, MDA-MB-231, and HepG2 cells hardly attached NMS-1286937 to the PMPC substrate within 1 h in both serum-containing and serum-free media. Conversely, these cells attached to the PBA, PTHFA, PMEA, PMe3A, and PMe2A substrates even in serum-free medium. These results indicate that HT-1080, MDA-MB-231, and HepG2 cells possess the ability to attach to these polymer substrates via integrin-independent attachment without adsorbed proteins. Open in a separate window Fig 6 Integrin-independent attachment.(A) HT-1080, (B) MDA-MB-231, and HepG2 were incubated with the substrates in either serum-free (FBS(-)) or serum-containing (FBS(+)) media for 1 h. The data represent the means SD (n = 3). *: < 0.05, ***: < 0.005 < 0.05 was amplified to normalize the expression of the genes of interest in the sample NMS-1286937 for each experiment. The PCR products were analyzed via 1% agarose gel electrophoresis. Table 1 Primer sequences for semi-quantitative RT-PCR analysis was designed according to Tuli et al. . and were designed in our laboratory. 4.8. Cell enrichment test HT-1080 and HepG2 cells were labeled via incubation with 10 M CellTracker Green (Life Technologies, Carlsbad, CA) and CellTracker Orange (Life Technologies) for 30 min at 37C. After washing, equal amounts of HT-1080 and HepG2 cells were mixed and then seeded at a total cell density of 5 104 cells/cm2. The non-attached cells were removed from the culture by washing twice with PBS after 1 h. The attached cells were fixed with 4% paraformaldehyde for 10 min at 37C. The attached cells were counted in three randomly selected fields using a confocal laser scanning microscope. 4.9. Statistical analysis All data are expressed as the means SD. The significance of the differences between two samples was determined by an unpaired Students < 0.05 were considered to be statistically significant. Supporting Information S1 FileSupporting figures and table. (Table A) Water content in hydrated PMEA-analogous polymers (wt%). (Figure A) Focal adhesion formation of MDA-MB-231 on PMEA-analogous polymer substrates after 1 day. (Figure B) Focal adhesion formation of HepG2 on the PMEA-analogous polymer substrates after 1 day. (Figure C) Relationship between intermediate water content and protein adsorption. (Figure D) Relationship between intermediate water contents and cell attachment. (Figure E) Chemical structure of PMEA analogous polymers. (DOC) Click here for additional data file.(2.2M, doc) Funding Statement This work was supported by the Funding Program for the Next Generation World-Leading Researchers (NEXT Program) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan and a Grant-in-Aid for Young Scientists (A) (26702016) from MEXT, Japan. Data Availability All relevant data are within the paper and its Supporting Information files..