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. Author manuscript; available in PMC: 2010 May 3.
Published in final edited form as: J Cell Biochem. 2005 Aug 15;95(6):1108–1119. doi: 10.1002/jcb.20473

Fig. 1.

Fig. 1

A: Western blot analysis of α-SMA expression after different TGF-β1 treatments. Subconfluent human gingival fibroblasts (HGFs) were stimulated with 0 (open bars), 0.25, 0.5, or 1 ng/ml TGF-β1 (gray bars) for 30 h. Whole cell lysates were equally loaded onto a 7.5% SDS–PAGE and α-SMA expression was determined. Densitometric analysis showed that TGF-β1 up-regulated α-SMA protein expression in a dose dependent manner. B: Subconfluent HGFs were pretreated with 0, 0.01, 0.1, or 1 mM nicotine (black bars) for 2 h and then 0.5 ng/ml TGF-β1 for 30 h. Duplicate samples were equally loaded and α-SMA detected on Western blots. TGFβ (TGF) (gray bar) had twofold more α-SMA than control untreated cells (NT open bar) and similar amounts as 0.01 or 0.1 mM nicotine treated cells (black bars). One millimolar of nicotine inhibited TGF-β1 induced α-SMA expression to below NT cell levels. C: HGFs were pre-treated with 1 mM nicotine (black bars) for 2 h before stimulation with 0.25, 0.5, or 1 ng/ml TGF-β1 (gray bars). Equal amounts of cell lysate were used for α-SMA Western blot analysis. Densitometric analysis showed that nicotine down-regulated TGF-β1-induced α-SMA expression at all concentrations of TGFβ. D: HGFs were pre-treated with or without differentiation concentrations of nicotine (black bars) and then treated with 0.5 ng/ml TGF-β1 as described for the Western blot analysis. α-SMA mRNA expression level was determined by using specific primers. The expression level of α-SMA mRNA in control untreated cells (NT open bar) was set at 1. TGF-β1 treatment increased the α-SMA mRNA expression 3.5-fold (gray bar). The cells pre-treated with 0.01 or 0.1 mM nicotine still had an increase of 3.1 or 3.2-fold respectively compared to non-treated controls. However, the cells pre-treated with 1 mM nicotine decreased the α-SMA mRNA expression levels to 0.6 compared to non-treated controls. Error bars equal the standard deviation between three experiments. E: TGFβ had little effect on β-actin mRNA and 0.01 mM nicotine was similar to TGFβ treated cells. Nicotine treatment at 0.1 and 1 mM decreased β-actin mRNA slightly.