Fig.1 Dioscin attenuates renal pathological changes and collagen deposition in UUO mice A: representative photomicrographs of HE staining of kidney tissues from each group on day 7 after UUO operation (bar=50 μm). Representative photomicrographs of Masson's trichrome staining of kidney tissues from each group (bar=50 μm). Blue areas indicate collagen deposition. B: Left: scoring of renal tubular damage assessed by loss of the brush border, tubular dilation, inflammatory cell infiltration degree according to random HE-stained kidney cortical region sections. Right: scoring of Masson's trichrome staining assessed by percentage of blue-stained collagen area in each selected random cortical region field ($ \overline{x} $±s, n=5). cP<0.01, compared with UUO group.

Fig.2 Effect of Dioscin on the expression of EMT-related markers in kidneys of UUO mice A: representative images of immunohistochemical staining for α-SMA and FN in renal tissues (bar=50 μm). B: representative Western blot images showing the protein expression levels of α-SMA, Vimentin and E-cadherin in renal tissues. C: quantitative analysis of α-SMA, Vimentin and E-cadherin protein expression. D: mRNA relative expression levels of FN, α-SMA, and E-cadherin in renal tissues detected by RT-PCR ($ \overline{x} $±s, n=3). bP<0.05, cP<0.01, compared with UUO group.

Fig.3 Effect of Dioscin on the expression of Akt/GSK-3β pathway proteins in kidneys of UUO mice A: representative Western blot images showing the protein expression levels of Akt, p-Akt (Ser473), GSK-3β, and p-GSK-3β (Ser9) in renal tissues. B: quantitative analysis of p-Akt, Akt, p-GSK-3β and GSK-3β protein expression and the ratios of p-Akt/Akt and p-GSK-3β/GSK-3β ($ \overline{x} $±s, n=3). bP<0.05, cP<0.01, compared with UUO group.

Fig.4 The Akt phosphorylation agonist SC79 aggravates TGF-β1-induced EMT-like phenotypic changes in HK-2 cells A: representative Western blot images showing the protein expression levels of α-SMA and Vimentin in HK-2 cells from each group. B: quantitative analysis of α-SMA and Vimentin protein expression. C: representative Western blot images showing the protein expression levels of Akt, p-Akt, GSK-3β, and p-GSK-3β. D: quantitative analysis of p-Akt, Akt, p-GSK-3β and GSK-3β protein expression and the ratios of p-Akt/Akt and p-GSK-3β/GSK-3β. E: mRNA relative expression levels of α-SMA, Vimentin, and E-cadherin detected by RT-PCR ($ \overline{x} $±s, n=3). bP<0.05, cP<0.01, compared with the TGF-β1 group.

Fig.5 Dis inhibits TGF-β1-induced EMT-like phenotypic changes and Akt/GSK-3β pathway activation in HK-2 cells A: cell viability of HK-2 cells treated with different concentrations of Dis for 24, 48, and 72 h, assessed by CCK-8 assay ($ \overline{x} $±s, n=8). cP<0.01, compared with the 0 μmol/L group. B: representative Western blot images showing the protein expression levels of α-SMA and Vimentin in HK-2 cells from different treatment groups. C: quantitative analysis of α-SMA and Vimentin protein expression. D: mRNA relative expression levels of α-SMA and Vimentin detected by RT-PCR. E: immunofluorescence staining for Vimentin (green) and E-cadherin (green). Scale bar=25 μm. F: representative Western blot images showing the protein expression levels of Akt, p-Akt, GSK-3β, and p-GSK-3β. G: quantitative analysis of the ratios of p-Akt/Akt and p-GSK-3β/GSK-3β ($ \overline{x} $±s, n=3). bP<0.05, cP<0.01, compared with the TGF-β1 group.

Fig.6 Dis counteracted the effects of SC79 on Akt/GSK-3β pathway activation and EMT marker expression in HK-2 cells A: representative Western blot images showing the protein expression levels of α-SMA and Vimentin. B: quantitative analysis of α-SMA and Vimentin protein expression. C: representative Western blot images showing the protein expression levels of Akt, p-Akt, GSK-3β, and p-GSK-3β. D: representative Western blot images showing the protein expression levels of Akt, p-Akt, GSK-3β, and p-GSK-3β ($ \overline{x} $±s, n=3). bP<0.05, cP<0.01, compared with the TGF-β1+SC79 group.