Figure 4.

Recombinant Alix coated on the substratum partially rescues the defects of Alix-knockdown WI38 fibroblasts in cell spreading and fibronectin matrix assembly. (A) After control or Alix-knockdown WI38 cells were grown on GST- or GST–Alix-coated coverslips for 24 h, cells were observed under a phase-contrast microscope and images were taken at × 100 magnification. (B) After five fields of cells randomly photographed from each treatment in (A) were enlarged and printed, individual cell lengths were manually measured and the relative average cell length and s.e.m. among different fields (error bars) were calculated. Statistical analysis of the significance was performed by Student's t-tests. (C) After control or Alix-knockdown WI38 cells were grown on GST- or GST–Alix-coated coverslips for 48 h, total proteins or DOC-soluble and DOC-insoluble proteins were extracted. Whereas total proteins were immunoblotted with anti-Alix, anti-FN and anti-actin antibodies, DOC-soluble and DOC-insoluble proteins were immunoblotted with anti-fibronectin antibodies. (D) Control or Alix-knockdown WI38 cells grown on GST- or GST–Alix-coated coverslips for 48 h were immunostained with anti-FN antibodies (green) and counterstained with PI (red).