Phospho-Smad2/3 (Thr8) Antibody - #AF3367

Fig. 8. Neferine inhibited the TGF-β1/Smad2/3 signaling pathway in abdominal aortic tissues SHRs. IHC analysis was performed to determine the protein expression of TGF-β1 (A), Smad2/3 and (B) p-Smad2/3 (C). All micrographs were taken at 400 × magnification. Scale bar = 50 µm. Representative images are shown on the left panel and statistical graph on the right panel. Data were denoted as means ± standard deviations; # p 

Figure 3 BMP8B triggers SMAD2/3 signaling to suppress adipogenesis. (A,B) Analysis using immunoblotting and quantification was conducted to assess the protein levels of p-SMAD1/5/8, p-SMAD2/3, p-ERK1/2, p-p38 MAPK, and p-JNK in LV-Bmp8b. (C) A model of BMPs-associated signal transduction. (D) Quantification was performed to determine the luciferase reporter activity driven by BRE, which pCMV-Bmp8b cotransfected with pCMV-Alk2, pCMV-Alk3, pCMV-Bmpr2, pCMV-Acrv2a, respectively. (E) Quantification was performed to determine the luciferase reporter activity driven by CAGA, which pCMV-Bmp8b cotransfected with pCMV-Alk2, pCMV-Alk4, pCMV-Alk5, pCMV-Alk7, pCMV-Tgfβr2, pCMV-Acrv2a, and pCMV-Acrv2b, respectively. (F,G) In the presence of DMH1 or TP0427736 HCL, the cells were induced to differentiate into adipocytes. On Day 8, Oil Red O staining was performed (F). Quantification of lipid content after adipogenic differentiation (G). Scale bar = 20 µm. The symbols in the charts represent three biological replicates. The data were presented as mean ± SD and analyzed using one-way ANOVA (ns not significant, ** p < 0.01, *** p < 0.001).

Fig. 5 Bmp8a activates Smad2/3 signaling to inhibit adipocyte differentiation in 3T3-L1 cells. a–d Representative western blot analysis and quantification of changes in p-Smad1/5/8, p-Smad2/3, p-ERK1/2, p-p38 MAPK, and p-JNK expression in LV-bmp8a cells (a, b) or LV-Bmp8a cells (c, d). Protein expression levels were quantified using ImageJ software and normalized to the amount of total protein (n = 3). e, f Representative Oil Red O staining photographs of LV-bmp8a and LV-Bmp8a 3T3-L1 cells were induced to adipogenic in the presence of DMH1 or TP0427736 HCL, dimethylsulfoxide (DMSO) as a vehicle and subjected to OD492 quantifications (n = 3). Scale bar = 20 µm. g Schematic diagram of BMP8 mediated signal transduction. BMP8 can activate Smad1/5/8 signal transduction through the receptor complex formed by type I receptor ALK2, ALK3, or ALK6 and type II receptor ACVR2A or BMPR2. Meanwhile, BMP8 can also activate Smad2/3 signal transduction through the receptor complex formed by type I receptors ALK4 or ALK5 and type II receptors ACVR2A, ACVR2B, or TGFBR2. h Non-expression of mouse Alk6 gene in 3T3-L1 cells (n = 3). i The qPCR quantification of the type I receptor (Alk2, Alk3, Alk4, Alk5, Alk7) and type II receptor (Acvr2a, Acvr2b, Bmpr2, Tgrβr2) transcripts expressed in 3T3-L1 cells (n = 3). j, k Quantification of the activity of BRE-driven luciferase reporters with pCMV-bmp8a (j) or pCMV-Bmp8a (k) cotransfected with pCMV-Alk2, pCMV-Alk3, pCMV-Bmpr2, pCMV-Acrv2a, respectively (n = 3). Renilla luciferase was used as the internal control. l, m Quantification of the activity of CAGA-driven luciferase reporters with pCMV-bmp8a (l) or pCMV-Bmp8a (m) cotransfected with pCMV-Alk2, pCMV-Alk3, pCMV-Bmpr2, and pCMV-Acrv2a, respectively (n = 3). Renilla luciferase was used as the internal control. Data were representative of at least three independent experiments. Data were analyzed by One-way ANOVA and presented as mean ± SD

Figure 10 Molecular mechanism of Spp1 in regulating fibroblast behavior (A) Western blot analysis of the effects of knocking down or overexpressing Spp1 on the expression of Smad2/3, Col1a1, and α-SMA under TGF-β induced conditions. (B–D) Protein expression grayscale analysis to compare the expression and phosphorylation differences of the proteins. (E–H) Double immunofluorescence staining of P-Smad2/3 (red) and Col1a1 (green) or α-SMA (green) to validate whether Spp1 regulates Col1a1 and α-SMA expression through the phosphorylation of Smad2/3. Blue represents cell nuclei stained with DAPI. In all the images, compared to the sh-NC or oe-NC groups, ∗ represents p < 0.05, ∗∗ represents p < 0.01, and ∗∗∗ represents p < 0.001; bar equals 20 μm; each experiment was conducted three times with six replicates each time.

Figure 6. Aloin inhibited CCl4-induced liver fibrosis through TGF-β/Smad signaling pathway in vivo. (A) Western blotting analysis of TGF-β1 (B), Smad2, Smad3, P-smad2/3 (C), Smad7 (D), c-Jun (E), and GAPDH expression in liver tissue. (F) Immunohistochemistry staining of P-smad2/3 in the liver tissues (magnification, 10× and 20×). (G) Quantification of areas of positive staining using ImageJ software. The proportion of cells stained brown (positive expression) to the entire field of view was analyzed. In the experiment, we randomly selected more than 3 repetitions of different fields of view. Data are expressed as mean ± standard deviation. * P < 0.05 and *** P < 0.001 versus the control group; ## P < 0.01 and ### P < 0.001 versus the CCl4 group; ns: no significance.

Figure 6. Aloin inhibited CCl4-induced liver fibrosis through TGF-β/Smad signaling pathway in vivo. (A) Western blotting analysis of TGF-β1 (B), Smad2, Smad3, P-smad2/3 (C), Smad7 (D), c-Jun (E), and GAPDH expression in liver tissue. (F) Immunohistochemistry staining of P-smad2/3 in the liver tissues (magnification, 10× and 20×). (G) Quantification of areas of positive staining using ImageJ software. The proportion of cells stained brown (positive expression) to the entire field of view was analyzed. In the experiment, we randomly selected more than 3 repetitions of different fields of view. Data are expressed as mean ± standard deviation. * P < 0.05 and *** P < 0.001 versus the control group; ## P < 0.01 and ### P < 0.001 versus the CCl4 group; ns: no significance.

Supplementary Figure S8. ESCs-Exo treatment of FB in vitro activates TGFb and Akt signaling. (a‒c) Expression levels of TGFb1, SMAD2/3, and quantification. (d‒g) Expression levels of TGFb2, TGFb3, pSMAD2/3 (Thr8), and quantification. (h‒k) Expression levels of Akt and pAkt and quantification. n ¼ 3 biological replicates. Data are represented as mean SD; one-way ANOVA with Fisher’s posthoc test. *P < 0.05 versus control and #P < 0.05 versus FB-Exo. Akt, protein kinase B; ESC-Exo, epidermal stem cellederived exosome; FB, fibroblast; FB-Exo, fibroblast-derived exosome; pAkt, phosphorylated protein kinase B; pSMAD, phosphorylated SMAD.