Figure 8

Oxidative stress induces the expression and secretion of TGF-β1 and TGF-β2. (a–d) ECs were incubated in the absence (−) or presence (+) of 10 μM H₂O₂ for 24 (a, c) or 72 h (b, d), and mRNA expression of TGF-β1 (a, b) and TGF-β2 (c, d) was then measured by means of qPCR. Determinations were performed in at least triplicates, and the results are expressed normalized relative to 28S mRNA expression. Significant differences were assessed by Student’s t-test (Mann–Whitney). *, P<0.05, **, P<0.01 against untreated condition. Graph bars show the mean±s.d. (N=3–5). (e–h) ECs were incubated in the absence (−) or presence (+) of 10 μM H₂O₂ for 72 h, and the protein secretion of TGF-β1 (e, f) and TGF-β2 (g, h) was then measured in the supernatant. (e, g) Representative images of western blot experiments performed for detection of TGF-β1 (e) and TGF-β2 (g) secretion. (f and h) Densitometric analyses of the experiments shown in (e and g), respectively. Protein levels were normalized against tubulin, and data are expressed relative to the untreated condition. Significant differences were assessed by Student’s t-test (Mann–Whitney). *, P<0.05 and **, P<0.01 against untreated condition. Graph bars show the mean±s.d. (N=3–4). (i–l) Cytosol (CF) and nuclear (NF) fractions were obtained from non-treated ECs and EC treated in the absence (−) or presence (+, 10 μM H₂O₂) of H₂O₂ for 72 h, with or without 0.5 μM SB431542 for 72 h, and expression of NF-κB p65 was analyzed. (i, k) Representative images from western blot experiments performed for detection of NF-κB p65 in the CF (i) and NF (k) from non-treated ECs and EC treated with H₂O₂ in the presence or absence of SB431542. (j and l) Densitometric analyses of the experiments shown in (i and k), respectively. Protein levels were normalized against tubulin and histone H3 for CF and NF, respectively, and data are expressed relative to non-treated condition (N=3).