Fig. 2
Effect of SB203580 on TGF-β1-induced up-regulation of ET-1 in A549 cells. Confluent A549 cells were treated with 10 ng/mL TGF-β1 or 10 μM SB203580 respectively while cells were pre-treated with 10 μM SB203580 for 60 min before 10 ng/mL TGF-β1 stimulation for SB203580 + TGF-β1 group. The samples were analyzed by real-time qPCR, ELISA and western blotting. The results of real-time qPCR were shown in Fig. 2a. The relative ET-1 mRNA level (normalized to GAPDH mRNA) in TGF-β1 group was significantly increased than those in control group (*p < 0.05). The relative ET-1 mRNA level in SB203580 + TGF-β1 group was decreased compared to TGF-β1 group (#p < 0.05). The results of ELISA assay in Fig. 2b also demonstrated the same results that TGF-β1 treatment increased the levels of ET-1 protein expression (*p < 0.05) and the combination of SB203580 could suppressed this effects (#p < 0.05). All the results are presented as means ± SD of 3 independent experiments performed in triplicate. Western blotting shows that TGF-β1 made the protein phosphorylation increased of NF-kB p65, JNK/SAPK (Fig. 2c) but the inhibitors of these pathways could not suppress the ET-1 protein expression according to the results of ELISA. TGF-β1 made the protein phosphorylation increased of p38 and SB203580 can effectively inhibit the expression of phospho-p38 (Fig. 2d). (C, control group; TGF, TGF-β1 group; SB, SB203580 group; SB + TGF, SB203580 + TGF-β1 group)