Phospho-Smad1/5/9 (Ser463+Ser465) Antibody - #AF8313

Fig. 6 The ERK and BMP2 signaling pathway is activated by high extracellular Mg2+ in DPSCs during odontogenic differentiation. a ERK phosphorylation was significantly enhanced in DPSCs treated with 1 mM, 5 mM, and 10 mM Mg2+ compared with the 0 mM Mg2+ group, but p38 and JNK phosphorylation amounts were unchanged. b ERK phosphorylation was reduced by 2-APB. c Consistently, the protein levels of BMP2, BMPR1, and phosphorylated Smad1/5/9 were significantly increased in DPSCs exposed to high extracellular Mg2+. d BMP2, BMPR1, and phosphorylated Smad1/5/9 protein amounts were decreased by 2-APB

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 6 Effects of CREB and BMP/Smad signaling on the expression of Wnt10b in C3H10T1/2 cells. (A) Western blot assay shows the effect of PTEN and/or BMP9 on CREB and phospho-CREB (p-CREB) in C3H10T1/2 cells. (B) Western blot assay shows the effect of PTEN knockdown and/or BMP9 on CREB and p-CREB in C3H10T1/2 cells. (C) Quantification of Western blot assay shows the effect of PTEN and/or BMP9 on p-CREB in C3H10T1/2 cells (**p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. groups treated with BMP9 only). (D) Quantification of Western blot assay shows effect of PTEN knockdown and/or BMP9 on p-CREB in C3H10T1/2 cells (**p < 0.01 vs. control; #p < 0.05 and ##p < 0.01 vs. groups treated with BMP9 only). (E) Western blot assay shows the effect of PTEN and/or BMP9 on Smad1/5/9 and p-Smad1/5/9 in C3H10T1/2 cells. (F) Western blot assay shows the effect of PTEN knockdown and/or BMP9 on Smad1/5/9 and p-Smad1/5/9 in C3H10T1/2 cells. (G) Quantification of Western blot assay shows the effect of PTEN and/or BMP9 on p-Smad1/5/9 in C3H10T1/2 cells (**p < 0.01 vs. control; ##p < 0.01 vs. groups treated with BMP9 only). (H) Quantification of Western blot assay shows effect of PTEN knockdown and/or BMP9 on p-Smad1/5/9 in C3H10T1/2 cells (**p < 0.01 vs. control; ##p < 0.01 vs. groups treated with BMP9 only). (I) IP assay shows the interaction between p-Smad1/5/9 and p-CREB in C3H10T1/2 cells. (J) IP assay shows the interaction between p-CREB and p-Smad1/5/9 in C3H10T1/2 cells. (K) ChIP assay shows the enrichment of p-CREB or p-Smad1/5/9 in the promoter region of Wnt10b C3H10T1/2 cells.