FIG. 3.

Inhibition of BTEB by antisense BTEB oligonucleotides significantly reduces acetaldehyde-induced αI(I) collagen mRNA levels. Serum-starved HSC were treated with or without acetaldehyde (100 μM) plus sense or antisense BTEB oligonucleotides at the indicated concentrations for 48 h. The medium was replaced once with fresh DMEM containing acetaldehyde and antisense or sense BTEB oligonucleotides. (A) To determine the optimal concentration of antisense BTEB oligonucleotides, whole-cell protein extracts (30 μg) were analyzed by Western blotting using a polyclonal anti-BTEB serum. (B) Compared to the BTEB sense oligonucleotides, the effectiveness of the BTEB antisense oligonucleotides at 50 μg/ml in blocking acetaldehyde-induced αI(I) collagen gene expression was confirmed in HSC transfected with the αI(I) collagen reporter P1.7/1.6 by CAT assays. Values presented here reflect the means ± standard deviations (n = 6). (C) A representative αI(I) collagen RPA gel is shown. Ten micrograms of total RNA from HSC treated with or without acetaldehyde (100 μM) plus sense or antisense BTEB oligonucleotides at 50 μg/ml were used. Upper arrow, αI(I) collagen mRNA; lower arrow, cyclophilin mRNA, as a control. (D) Quantitation of αI(I) collagen mRNA in an RPA (Fig. 3B) by computer-aided phosphorimaging densitometry. Loading variation was normalized by cyclophilin mRNA. Representative gels are shown.