Fig. 5. ORP3 regulates HEK293 cell adhesion, spreading and β1 integrin activity. (A) Effect of ORP3 overexpression and silencing on cell spreading. HEK293 cells were transfected with ORP3 cDNA, with the empty vector (Mock), or with ORP3-specific (siORP3) or control (siC2) siRNAs. NT, non-transfected cells. Trypsinized cells were plated on fibronectin-coated coverslips for 1 hour, fixed and stained with OG488P. The area covered by the cells was determined from confocal microscope images. The figure shows mean area (± s.e.m.) of cells analysed from three different coverslips in the same experiment. The total numbers of cells analyzed are indicated. The expression levels of ORP3 and β-actin were assayed by western blotting of 20 µg total cell protein (2 µg for the ORP3-transfected specimen). (B) Effect of ORP3 silencing on cell adhesion on substratum. HEK293 cells treated with siC2 or siORP3 were trypsinized, allowed to adhere on collagen substratum for 1 hour and the number of adherent cells was determined. (C) ORP3 modulates β1 integrin activity. The amount of active integrin in cells transfected with ORP3 cDNA (ORP3), empty plasmid (Mock), or ORP3 siRNA was determined by FACS analysis using P4G11 antibody, which recognizes active β1 integrin. The amount of active integrin was calculated relative to total β1 integrin (P5D2 antibody) expressed on the cell surface. (D) Rescue of the ORP3-silencing phenotype. HEK293 cells were transfected with empty plasmid and ORP3 siRNA (siORP3+mock) or ORP3 mutant cDNA resistant to the siRNA and ORP3 siRNA (siORP3+ORP3mut), followed by analysis of the amount of active integrin on the cell surface. Western blot panels verify successful ORP3 overexpression and silencing. The results represent mean ± s.e.m.; *P<0.05; **P<0.01; ***P<0.001. (E) R-Ras regulates integrins downstream of ORP3. HEK293 cells transiently transfected with empty plasmid and ORP3 siRNA (siORP3+mock) or ORP3 siRNA and R-Ras constructs, were studied for integrin activity as above, or were allowed to spread on fibronectin-coated coverslips for 120 minutes, fixed and stained as indicated. Scale bars, 10 µm.