Fig. 8. Reggie-1 influences cell spreading in a tyrosine-dependent manner. HeLa cells were transfected with various R1-EGFP constructs or reggie-1-specific siRNA duplex oligoribonucleotides and allowed to spread on fibronectin-coated glass chambers for 25 minutes. After washing, cells were fixed, stained with fluorochrome-coupled phalloidin and scored as non-spread (merely attached and round), half-spread (non-flattened with filopodial protrusions) or spread (flattened with lamellipodia). (A) Representative cells of the three spreading categories (phalloidin staining). (B) R1-EGFP and most of the Tyr mutants, including 7xYF, were capable of enhancing cell spreading compared with the EGFP control, whereas the soluble G2A and partly soluble Y27F mutants strongly inhibited spreading. However, Y24F, Y124F and Y163F mutants showed no enhancing or inhibiting effect on cell spreading and were comparable with EGFP, indicating a role for these phosphorylation sites of reggie-1 in cell spreading. (C) HeLa cells transfected either with reggie-1-specific siRNA duplex oligoribonucleotides or control were subjected to the same spreading assay. Compared with the control cells, the reggie-1 knockdown cells spread less efficiently on fibronectin. (D) A representative western blot shows the significant knockdown of reggie-1 (85% for R1S3 and 89% for R1S1 oligos) compared with cells treated with control siRNA. GAPDH was used as a loading control for the lysates.