Figure 5. Surface-bound fibronectin (FN)-growth factor-binding domains (GFBDs) contiguous with FN-central cell-binding domain (FNIII_8–11) prevented metabolic arrest, autophagy, and apoptosis of FN-null cells even in the absence of platelet-derived growth factor (PDGF).

FN-null fibroblasts were cultured at 4,000 cells per well in 96-well plates precoated with 0.125 µm intact FN or glutathione S-transferase (GST)-tagged FNIII_8–11, FNIII_1–11, or FNIII_8–v15 in DMEM for 4 hours, and then incubated with DMEM and 1% BSA with or without 30 ng ml⁻¹ PDGF-BB at 37 °C for times indicated. (a) Metabolic response over 4 hours as judged by XTT assay. (b) Total cellular light chain 3 (LC3), a component of autophagosomes, was detected by rabbit polyclonal antibodies using ELISA (Jager et al., 2004). (c) LC3-II, a translationally modified product of LC3 that binds to autophagosomes, was detected by a size shift on western blot analysis using a polyclonal antibody specific for LC3 (Zhang et al., 2007). Alpha-actin was probed as a housekeeping protein in all gels and showed no substantial difference among samples within a given gel. (d) Apoptosis was determined by the TUNEL assay for fragmented DNA manifested by bright yellow fluorescent spots on a background of red 4′,6-diamidino-2-phenylindole-stained nuclei. Percent apoptosis was determined by counting those cells positive for DNA fragmentation, dividing by total cell number, and multiplying by 100. Fifty cells were counted in each of three replicate plates. Data points indicate mean ±SD. In panels a and b, each data point was performed in quadruplicate and each panel is representative of at least three different experiments. The western blot in panel c is representative of two different experiments. OD, optical density.