Figure 9.

IGFBP-5’s effects are Egr-1-dependent and -independent. A: Fibronectin induction by IGFBP-5 is abolished in Egr-1 KO mouse fibroblasts. Fibroblasts were infected with Ad-IGFBP-5 (Ad5) or cAd. Fibronectin expression was analyzed by Western blot. B: Graphical summary from three independent experiments. Intensity of the bands on Western blot was analyzed using ImageJ and the ratio of fibronectin to GAPDH was calculated; *P < 0.05. C: Fibroblasts were infected with Ad-IGFBP-5, and cytoplasmic and nuclear fractions were extracted at 24 to 96 hours. Expression of Egr-1 (nuclear) and fibronectin (cytoplasmic) were examined by Western blot. Histone and GAPDH were used as loading controls in nuclear and cytoplasmic extracts, respectively. D: Fibroblasts were infected with Ad-IGFBP-5. Cytoplasmic and nuclear fractions were extracted after 72 hours. Expression of IGFBP-5 was analyzed by Western blot. Signal intensity was quantified using ImageJ. The number denotes ratio of IGFBP-5 in nuclear extract to that in cytoplasmic extract. E: Egr-1 KO lung fibroblasts were infected with Ad-IGFBP5, Ad-Egr-1, or both at a multiplicity of infection of 25 each. Extracellular matrix was harvested after 72 hours. Fibronectin deposition was assessed in the extracellular matrix using Western blot. Vitronectin served as a loading control.