Figure 4.

The p27 expression is low in IPF fibroblasts. A: Serum-starved control and IPF fibroblasts were cultured on polymerized collagen gels as a function of time, and p27 expression was measured by Western blot analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. B: IPF fibroblasts were infected with an adenovirus expressing WT FoxO3a (F3), DF, or empty vector (GFP), and FoxO3a, p-FoxO3a, p27, and GAPDH expression levels were measured at 24 hours. C: Top: FoxO3a^−/− cells were reconstituted with F3 or empty vector (GFP), and FoxO3a, p27, and GAPDH levels were measured at 24 hours. Bottom: DF was expressed in control fibroblasts, and FoxO3a and p27 expression levels were measured at 24 hours. GAPDH was used as a loading control. D: HA, DA, or empty vector (GFP) was overexpressed in IPF fibroblasts, and p27 expression was measured at 24 hours. GAPDH was used as a loading control. E: Serum-starved control fibroblasts in suspension culture were ligated with a β1 integrin–activating antibody (TS2/16) or an isotype control IgG antibody (3 μg/mL) for 1 hour. Cells were then collected and Western blot analysis was performed to measure p27 levels. GAPDH was used as a loading control.