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. 2014 Sep 4;9(9):e106554. doi: 10.1371/journal.pone.0106554

Figure 6. AMPKα1 silencing and compound C blocks the inhibitory effects of AICAR on TGF-β1-induced myofibroblast activation.

Figure 6

Cultured NRK-49F cells were transfected with a siRNA specific for AMPKα1 (10 nM) or a control siRNA for 24 h. After 24 h of incubation, the transfected cells were pre-incubated with or without AICAR (0.5 mM) for 30 mins. Then, these cells were stimulated with TGF-β1 (1 ng/mL) for 24 h before harvesting. (A) Culture supernatant and cell lysates were subject to immunoblot analysis with antibodies against collagen I, collagen IV, α-SMA, and tubulin. (B) Cultured NRK-49F cells were pre-incubated with compound C (1.0 mM, 2.5 mM, or 5.0 mM) for 30 mins. Then, these cells were stimulated with TGF-β1 (1 ng/mL) for 24 h before harvesting. Immunoblotting of the cell lysates, showed that the inhibitory effects of AICAR on α-SMA expression were decreased by the treatment with Compound C in a dose-dependent manner. Representative immunoblots from three experiments are shown. Each bar represents the mean ± S.E. of three independent experiments. *P<0.05 versus the control group; #P<0.05 versus the TGF-β1 (+/− mock) group; ##P<0.05 versus the TGF-β1 (+/− mock) + AICAR group. C: control, T: TGF-β1, M: mock, A: AICAR.