Immunofluorescence analysis of AMPK and Smad3 pathways.

NRK52E cells were treated with TGF-β1 (10 ng/ml) with or without HL156A (30 μM) for 24 hours. (A) Immunofluorescence staining showed expression of p-AMPK (red), p-Smad3 (red), E-cadherin (red), α-SMA (green), fibronectin (green), and type IV collagen (green). This revealed that HL156A, by stimulating p-AMPK, inhibited p-Smad3, α-SMA, fibronectin, and type IV collagen, which were all induced by TGF-β1 treatment. HL156A co-treatment also restored E-cadherin expression that was suppressed under TGF-β1 treatment. (B) Immunofluorescence analysis quantified using image J. Experiments were repeated three times. *P #P < 0.05 between TGF-β1 and TGF-β1 + HL156A.