Figure 5.

The importance of PDGFRα and suppression of p53 for RV-dependent cell proliferation and protection from apoptosis and senescence. A: RCFs (shGFP, shPDGFRα, shPDGFRα/shp53, and shp53) are seeded into a 24-well plate at a density of 5 × 10⁴ cells per well in DMEM plus 10% FBS. Six hours after the cells attach, the medium is changed to either 0.5-mL DMEM or rabbit vitreous (diluted 1:3 in DMEM). The media are replaced every day. The cells are counted with a hemocytometer on day 3. The mean ± SD of three independent experiments is shown. *P < 0.05 using a paired t-test. B: The RCFs in A are seeded into 60-mm dishes at a density of 100,000 cells per dish in DMEM plus 10% FBS. Six hours after the cells attach, the medium is changed to either 3-mL DMEM or rabbit vitreous (diluted 1:3 in DMEM). The media are replaced every day. On day 3, the cells are stained with fluorescein isothiocyanate–conjugated annexin V and propidium iodide (PI) in an apoptosis assay kit by following the manufacturer's instructions. Cells that are stained with annexin V and/or PI are detected and quantified by flow cytometry in Coulter Beckman XL. The mean ± SD of three independent experiments is shown. C: The RCFs in A are seeded into a 12-well plate at a density of 10,000 cells per well in DMEM plus 10% FBS. Six hours after the cells attach, the medium is changed to either 1-mL DMEM or rabbit vitreous (diluted 1:3 in DMEM). The media are replaced every day. On day 3, the β-galactosidase activity is measured as outlined in the manufacturer's instructions. Both stained and unstained cells are counted and photographed under an inverted microscope. The mean ± SD of three independent experiments is shown. *P < 0.05 using a paired t-test. NS, not significant. Scale bar = 50 μm.