Figure 4

Ha-Ras up-regulates collagen I independently of autocrine transforming growth factor-β (TGFβ). (A) Luciferase activity (expressed as fold induction over control sample) of the human pro-α2 (I) collagen gene (COL1A2) promoter co-transfected with the ca-Ras expressing plasmid in quiescent human dermal fibroblast (hDF) cultured in the presence or absence of TGFβ neutralizing antibody (2 and 10 μg/mL); cells stimulated with recombinant TGFβ1 (2 ng/mL) served as a positive control. Luciferase values represent the average of three independent transfections each performed in duplicate and error bars signify ± standard deviation and asterisks indicate statistically significant differences (P < 0.05). (B) Collagen I immunoblots of protein extracts (n = 3 per each sample) from mouse dermal fibroblast (mDF) cultures infected with Ha-Ras or control lentiviruses and cultured in the presence or absence of TGFβ neutralizing antibody (10 μg/mL) for the indicated times. (C) pSmad3 immunoblots of protein extracts (n = 3 per each sample) from mDF cultures stimulated with recombinant TGFβ1 (2 ng/mL) in the presence or absence of neutralizing TGFβ antibodies (10 μg/mL). In the last two panels, bar graphs summarize the ratio of pSmad3 or collagen I relative to the loading control glyceraldehyde 3-phosphate dehydrogenase (GADPH) in the various experimental samples; asterisks indicate statistically significant differences (P < 0.05).