FIGURE 7.

Cell attachment to collagen promotes caspase-3-dependent cleavage of FoxO3a. A, RT-PCR was carried out with primers designed for FoxO3a as described under “Materials and Methods.” Actin was used as a loading control. B, upper panel, serum-starved human lung fibroblasts were pretreated with caspase-3 inhibitor (30 nm) for 60 min. Cells were then attached to type I collagen as a function of time. FoxO3a levels were measured. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. DMSO, control. Lower panel, serum-starved human lung fibroblasts were pretreated with different doses of caspase-3 inhibitor (3 to 300 nm) for 60 min. Cells were then attached to type I collagen for 30 min. FoxO3a levels were then measured. GAPDH was used as a loading control. DMSO, control. C, serum-starved human lung fibroblasts were pretreated with β1-integrin blocking antibody (1 μg/ml) or isotype control antibody IgG (1 μg/ml) for 45 min. Cells were then attached to type I collagen (100 μg/ml) for 60 min. Caspase-3 activity assay was performed as described under “Materials and Methods.”