NaHS attenuates the TGF-β1 induced EMT via Wnt/catenin pathway.

<p>(A) β-catenin knockdown efficiency by siRNA in HK-2 cells. The cells were transfected with β-catenin siRNA or control siRNA for 6 hours and the culture medium was replaced by fresh medium. Western blot assay was used to detect the expression level of non-phosphorylated β-catenin. (B) After transfected with β-catenin siRNA or control siRNA for 6 hours, HK-2 cells were incubated with NaHS (100μM) for 12 hours and then with TGF-β1 for the following 36 hours. The expression level of fibronectin, E-cadherin, TβR I and α-SMA were examined by western blot assay. (C) Graphical representation of the relative quantification for the protein level. The relative values were calculated by the density of targeted proteins vs GAPDH (%). The values of mean ± SEM (n = 3) were gained from relative abundance quantified by densitometry and normalized to GAPDH. ^#P<0.05 vs. control group, *P<0.05 vs TGF-β1 group. One-way ANOVA followed by Tukey’s multiple comparison test.</p

NaHS increased the Cystathionine β-synthase (CBS) expression in HK-2 cells.

<p>Pretreatment by the several concentrations of NaHS is given to HK-2 cells for 12 hours and then TGF-β1 was given to the cells for the following 36 hours. (A) NaHS increased the CBS expression level in a concentration‑dependent manner in TGF-β1-stimulated HK-2 cells. (B) Graphical representation of the relative quantification for CBS. The relative values were calculated by the density of of CBS vs β-actin (%). The values of mean ± SEM (n = 3) were gained from relative abundance quantified by densitometry and normalized to β-actin. ^#P<0.05 vs. control group, *P<0.05 vs TGF-β1 group. One-way ANOVA followed by Tukey’s multiple comparison test.</p

NaHS attenuated TGF-β1-stimulated EMT in HK-2 cells.

<p>Pretreatment by the several concentrations of NaHS is given to HK-2 cells for 12 hours and then TGF-β1 was given to the cells for the following 36 hours. (A) The influence of 400μM NaHS on the morphological change of HK-2 cells (B) Western blot assay for fibronectin, E-cadherin and α-SMA expressions in HK-2 cells. (C) Graphical representation of the relative quantification for fibronectin, E-cadherin and α-SMA. The relative values were calculated by the density of fibronectin, E-cadherin and α-SMA vs GAPDH (%). The values of mean ± SEM (n = 3) were gained from relative abundance quantified by densitometry and normalized to GAPDH. ^#P<0.05 vs. control group, *P<0.05 vs TGF-β1 group. One-way ANOVA followed by Tukey’s multiple comparison test.</p

NaHS decreased the TGF-β1-stimulated overexpression of TGF-β receptor type II (TβR II) and TGF-β receptor type I (TβR I).

<p>Pretreatment by the several concentrations of NaHS is given to HK-2 cells for 12 hours and then TGF-β1 was given to the cells for the following 36 hours. (A) NaHS decreased both TβR I and TβR II overexpression stimulated by TGF-β1 in a concentration-dependent manner in HK-2 cells. (B) Graphical representation of the relative quantification for TβR I and TβR II. The relative values were calculated by the density of TβR I and TβR II vs GAPDH (%). The values of mean ± SEM (n = 3) were gained from relative abundance quantified by densitometry and normalized to GAPDH. ^#P<0.05 vs. control group, *P<0.05 vs TGF-β1 group. One-way ANOVA followed by Tukey’s multiple comparison test.</p

The anti-EMT effect of NaHS on HK-2 cells was blocked by the inhibitors of ERK1/2 and β-catenin.

<p>(A) The expression of non-phosphorylated β-catenin under the condition of ERK signal was blocked. ERK1/2 inhibitor U0126 (10μM) was given to HK-2 cells for 1 hour and then cells were incubated with TGF-β1 for 36 hours. (B) Effects of NaHS (100μM) on the expression of fibronectin, E-cadherin,TβR I and α-SMA in the presence of TGF-β1 and U0126 or the β-catenin inhibitors XAV-939. Pretreatment by NaHS (100μM) is given to HK-2 cells for 12 hours and then U0126 (10μM) or XAV-939 (10μM) were given to the cells for 1 hour before they were incubated with TGF-β1 for the following 36 hours. (C) Graphical representation of the relative quantification for the protein level. The relative values were calculated by the density of targeted proteins vs GAPDH (%). The values of mean ± SEM (n = 3) were gained from relative abundance quantified by densitometry and normalized to GAPDH. ^#P<0.05 vs. control group, *P<0.05 vs TGF-β1 group. One-way ANOVA followed by Tukey’s multiple comparison test.</p

NaHS decreased TGF-β1-stimulated overexpression of non-phosphorylated β-catenin.

<p>HK-2 cells were pretreated with NaHS at the indicated doses for 12 hours and then they were treated by TGF-β1 for the following 36 hours. (A) Western blot assay for β-catenin and non-phosphorylated β-catenin expression in HK-2 cells. (B) Graphical representation of the relative quantification for β-catenin and non-phosphorylated β-catenin. The relative values were calculated by the density of β-catenin and non-phosphorylated β-catenin vs GAPDH (%). The values of mean ± SEM (n = 3) were gained from relative abundance quantified by densitometry and normalized to GAPDH. ^#P<0.05 vs. control group, *P<0.05 vs TGF-β1 group. One-way ANOVA followed by Tukey’s multiple comparison test.</p

NaHS decreased TGF-β1-stimulated overexpression of phosphorylated ERK1/2.

<p>(A) HK-2 cells were incubated with TGF-β1 for several specific time periods, and the phosphorylated ERK1/2 was detected by western blot assay. (B) Western blot assay for phospho-ERK1/2 expression in HK-2 cells. Pretreatment by the several concentrations of NaHS is given to HK-2 cells for 12 hours and then TGF-β1 was given to the cells for 30 minutes at which time the phosphorylated ERK1/2 express the most (C) Graphical representation of the relative quantification for phospho-ERK1/2. The relative values were calculated by the density of phospho-ERK1/2 vs GAPDH (%). The values of mean ± SEM (n = 3) were gained from relative abundance quantified by densitometry and normalized to GAPDH. ^#P<0.05 vs. control group, *P<0.05 vs TGF-β1 group. One-way ANOVA followed by Tukey’s multiple comparison test.</p