Figure 1.
Effects of curcumin on morphology and the expression of α-SMA and PAI-1 and E-caherin in TGF-β1-induced HK-2 cells.
HK-2 cells were incubated with curcumin at the indicated dose for 12 hours followed by treatment with TGF-β1 for 36 hours. (A) Effects of 10 μM of curcumin on morphology of HK-2 cells (B) Western blotting analyses for α-SMA, E-caherin and PAI-1 expression in HK-2 cells. (C) Graphical presentation of the relative expression of α-SMA, E-caherin and PAI-1. The values were represented as the density of α-SMA, E-caherin or PAI-1 vs β-actin (%). The mean ± SD (n = 3) of values were obtained from densitometric analysis of all individual experiments. *p<0.05 vs TGF-β1 alone.
Figure 2.
Effects of curicumin on TGF-β1-induced TβR I and TβR II expression.
HK-2 cells were incubated with curcumin at the indicated dose for 12 hours followed by treatment with TGF-β1 for 36 hours. (A) Western blotting analyses for TβR I and TβR II expression in HK-2 cells. (B) Graphical presentation of the relative expression of TβR I and TβR II. The values were represented as the density of TβR I or TβR II vs β-actin (%). The mean ± SD (n = 3) of values were obtained from densitometric analysis of all individual experiments. #P<0.05 vs. control alone, *P<0.05 vs TGF-β1 alone.
Figure 3.
Effects of curcumin on TGF-β1-induced Smad2 and Smad3 phosphorylation in HK-2 cells.
(A) HK-2 cells were stimulates with TGF-β1 (2.5 ng/ml ) for the indicated times, and the phosphorylation of Smad2 and Smad3 were analyzed by western blotting. (B) Western blotting analyses for p-Smad2 and p-Smad3 expression in HK-2 cells. HK-2 cells were incubated with curcumin at the indicated doses for 12 hours followed by treatment with TGF-β1 for 30 minutes.
Figure 4.
Effects of curcumin on TGF-β1-induced ERK phosphorylation in HK-2 cells.
(A) HK-2 cells were stimulates with TGF-β1 (2.5 ng/ml ) for the indicated times, and the phosphorylation of ERK was analyzed by western blotting. (B) Western blotting analyses for p-ERK expression in HK-2 cells. HK-2 cells were incubated with curcumin at the indicated doses for 12 hours followed by treatment with TGF-β1 for 30 minutes. (C) Graphical presentation of the relative expression of pERK. The values were represented as the density of p-ERK vs total ERK (%). The mean ± SD (n = 3) of values were obtained from densitometric analysis of all individual experiments. #P<0.05 vs. control alone, *P<0.05 vs TGF-β1 alone.
Figure 5.
Curcumin reduced TGF-β1-induced PPARγ phosphorylation and reversed the reduction of PPARγ.
HK-2 cells were incubated with curcumin at the indicated dose for 12 hours or rosiglitazone for 1 hour followed by treatment with TGF-β1 for 36 hours. (A) Western blotting analyses for PPARγ and p-PPARγ expression in HK-2 cells. (B) Graphical presentation of the relative expression of PPARγ and p-PPARγ. The values were represented as the density of PPARγ and p-PPARγ vs β-actin (%). The mean ± SD (n = 3) of values were obtained from densitometric analysis of all individual experiments. #P<0.05 vs control alone, *P<0.05 vs TGF-β1 alone.
Figure 6.
Effects of curcumin on nuclear translocation of PPARγ.
HK-2 cells were pretreated with curcumin (10 μM) for 12 hours (C) or rosiglitazone (10 μM) for 1 hour (D) following by incubated with TGF-β1 for 36 hours. Red for PPARγ.
Figure 7.
ERK1/2 inhibitor U0126 and PPARγ inhibitor BADGE blocked the effects of curcumin on TGF-β1-induced EMT in HK-2 cells.
(A) Effects of U0126, BADGE on the expression of α-SMA, PAI-1, E-cadherin, TβR I and TβR II with or without TGF-β1. HK-2 cells were pretreated with curcumin (10 μM) for 12 hours followed by treatment with U0126 (10 μM) or BADGE (10 μM) for 36 hours or HK-2 cells were pretreated with U0126 (10 μM) or BADGE (10 μM) for 1 hour before addition of TGF-β1 (2.5 ng/ml) for 36 hours. (B) Effects of curcmin on the expression of α-SMA, PAI-1, E-cadherin, TβR I and TβR II with TGF-β1 and U0126 or BADGE. HK-2 cells were pretreated with curcumin (10 μM) for 12 hours at indicated dose followed by treatment with U0126 (10 μM) or BADGE (10 μM) for 1 hour before addition of TGF-β1 (2.5 ng/ml) for 36 hours.
Figure 8.
Effect of curcumin on TGF-β1-induced EMT in HK-2 cells is dependent on PPARγ.
(A) Knockdown of PPARγ in HK-2 cells. HK-2 cells were infected with PPARγ-specific shRNA or scrambled shRNA for 3 hours and then changed the medium. The expression of PPARγ was examined by western blotting. (B) After infected with PPARγ-specific shRNA or scrambled shRNA for 3 hours, HK-2 cells were treated with curcumin (10 μM) for 12 hours at indicated dose followed by treatment with TGF-β1 (2.5 ng/ml) for 36 hours. Expression of α-SMA, PAI-1, E-cadherin, TβR II and TβR I were detected by western blotting.
Figure 9.
Crosstalk between ERK and PPARγ signaling.
(A) Effect of blocking PPARγ signaling by pharmacological inhibitor on phoshorylation of ERK. HK-2 cells were pretreated with BADGE (10 μM) 1 hour before addition of TGF-β1 (2.5 ng/ml) for 30 minutes and the expression of p-ERK were analyzed by western blotting. (B) Effect of knockdown of PPARγ at cellular level on phoshorylation of ERK. HK-2 cells infected with PPARγ-specific shRNA or scrambled shRNA were treated TGF-β1 (2.5 ng/ml) for 30 minutes and the expression of p-ERK were analyzed by western blotting. (C) Effect of blocking ERK signaling on phoshorylation of PPARγ. HK-2 cells were pretreated with U0126 (10 μM) for 1 hour before addition of TGF-β1 (2.5 ng/ml) for 36 hours and the expression of PPARγ were analyzed by western blotting.
Figure 10.
Effects of curcumin on TGF-β1-induced EMT in NRK-52E cells.
NRK-52E cells were incubated with curcumin at the indicated dose for 12 hours followed by treatment with TGF-β1 for 36 hours(PAI-1, p-PPARγ and PPARγ) or 30 min(P-Smad2 and P-Smad3). (A) Western blotting analysis for PAI-1 expression. (B) Western blotting analysis for p-Smad2 and p-Smad3 expression. (C) Western blotting analysis for PPARγ and p-PPARγ expression.