Fig 2: The SMAD pathway is responsible for HaCaT cell autophagy induction by hBMSC-derived TGF-ß1. a Western blotting and b quantitative analysis were used to analyze the expression levesl of SMAD2, p-SMAD2 and SMAD4 in HaCaT cells subjected to CM from control hy-hBMSCs and siTGF-ß1 hy-hBMSCs for 24 h. c Representative fluorescence images of SMAD4-stained HaCaT cells after 24 h of treatment with different CM. Bar, 20 µm. d Western blotting and e quantitative analysis were used to examine the proportion of SMAD expression in the nucleus and cytoplasm of HaCaT cells subjected to 24 h of different CM. f Western blotting and g quantitative analysis were used to measure the protein level of SMAD2 to evaluate the efficiency of siSMAD2 in HaCaT cells at 24 h after hyCM administration. h Western blotting and i quantitative analysis were used to analyze the expression levels of LC3B-I/II, BECN1 and SQSTM1 in control HaCaT cells and siSMAD2 HaCaT cells at 24 h after hyCM administration. Mean ± SEM. n = 3. *P < 0.05, **P < 0.01

Fig 3: AKT/mTOR-signaling-pathway-mediated autophagy is required for the ASIV-induced alleviation of EMT and G2/M arrest in vitro. (a) Protein expressions of p-mTOR, t-mTOR, p-ULK1, t-ULK1, ATG7, SQSTM1, Beclin1, and LC3 I/II in HK-2 cells using Western blotting. (b) Quantitative analysis of the phosphorylation levels of mTOR and ULK1 and the relative protein expressions of ATG7, SQSTM1, Beclin1, and LC3 II/LC3 I. (c) Protein expressions of fibronectin, vimentin, and a-SMA in HK-2 cells using Western blotting. (d) Quantitative analysis of the relative protein expressions of fibronectin, vimentin, and a-SMA. (e) Protein expression of p-p53ser15, p53, p21, and p-H3ser10 in HK-2 cells using Western blotting. (f) Quantitative analysis of the phosphorylation level of p53 and the relative protein expressions of p21 and p-H3ser10. Representative images of immunofluorescence staining for (g) fibronectin, (h) F-actin, (i) Ki67 (red)/p-H3 (green), and (j) LC3 I/II in the HK-2 cells. Data are presented as mean ± SEM (n = 3, 100 µm scale bar). One-way ANOVA was followed by Dunnett’s multiple comparisons test. ### p < 0.001 vs. the control group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the TGF-ß1 group; ^ p < 0.05, ^^ p < 0.01, ^^^ p < 0.001 vs. the TGF-ß1 + ASIV group.

Fig 4: ALDH2 siRNA blocks the beneficial effects of ASIV on renal fibrosis in vitro. (a) Protein expression of ALDH2 in HK-2 cells using Western blotting. (b) Quantitative analysis of the relative protein expression of ALDH2. (c) Protein expression of fibronectin, vimentin, and a-SMA in HK-2 cells using Western blotting. (d) Quantitative analysis of the relative protein expressions of fibronectin, vimentin, and a-SMA. (e) Protein expression of p-p53ser15, p53, p21, and p-H3ser10 in HK-2 cells using Western blotting. (f) Quantitative analysis of the relative protein expressions of p-p53/t-p53, p21, and p-H3ser10 in HK-2 cells of each group using Western blotting. (g) Protein expression of p-AKT, t-AKT, p-mTOR, t-mTOR, ATG7, SQSTM1, Beclin1, and LC3 I/II in HK-2 cells of each group using Western blotting. (h) Quantitative analysis of the phosphorylation levels of AKT and mTOR and the relative protein expressions of ATG7, SQSTM1, Beclin1, and LC3 II/LC3 I. Representative image of immunofluorescence staining for (i) fibronectin, (j) F-actin, (k) Ki67 (red)/p-H3 (green), and (l) LC3 I/II in the HK-2 cells. Data are presented as mean ± SEM (n = 3, 100 µm scale bar). One-way ANOVA was followed by Dunnett’s multiple comparisons test. ## p < 0.01, ### p < 0.001 vs. the control group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the TGF-ß1 group; ^ p < 0.05, ^^ p < 0.01, ^^^ p < 0.001 vs. the TGF-ß1 + ASIV group.

Fig 5: ASIV promotes ALDH2 expression, inhibits autophagy, and activates AKT/mTOR signaling pathway in vivo. (a) ALDH2 mRNA level in the renal tissues using qRT-PCR for each group. (b) Protein expression of ALDH2 in the renal tissues of each group using Western blotting. (c) Quantitative analysis of the relative protein expression of ALDH2 in the renal tissues. (d) Representative images of immunohistochemistry staining for ALDH2 in the rat kidneys (n = 6, 100 µm scale bar). (e) Relative gene expressions of Mtor, Map1lc3b, Becn1, and Atg7 in the renal tissues of each group (n = 6). (f) Protein expressions of p-AKT, t-AKT, p-mTOR, t-mTOR, ATG7, SQSTM1, Beclin1, and LC3 I/II in the renal tissues of each group using Western blotting. (g) Quantitative analysis of the phosphorylation levels of AKT and mTOR in the renal tissues. (h) Quantitative analysis of the relative protein expressions of ATG7, SQSTM1, Beclin1, and LC3 II/LC3 I in the renal tissues. (i) Representative images of immunofluorescence staining for LC3 I/II/a-tubulin in the rat kidneys (n = 6, 100 µm scale bar). Data are presented as mean ± SEM. One-way ANOVA was followed by Dunnett’s multiple comparisons test. ### p < 0.001 vs. the control group, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. the adenine group.