Figure 3.

Induction of FN matrix assembly correlates with down-regulation of Raf-1 protein levels. Ras-GTP levels in HT-1080 cells were determined in 2D or 3D culture using a commercially available assay. (A) No difference in active or total Ras was noted. (B) Immunoblot analysis of Raf-1 expression in lysates generated from 3D cultures show a marked decrease in Raf-1 expression compared with cells cultured as monolayers. Raf-1 expression was normalized against the expression of GAPDH. (C) This decrease was quantified by densitometric analysis of five separate experiments comparing Raf-1 expression as a function of culture condition. GAPDH was used to normalize the data. An unpaired Student’s t test detected a significant difference (p < 0.05) in Raf-1 expression between cells grown as 2D and 3D cultures. (D) Transition to 3D culture induces FN matrix assembly in the rat prostatic carcinoma cell line MAT-LyLu. (E) This correlates with a decrease in Raf-1 expression in these cells.