Fig 1: BMP8B represses PPARγ transcription via SMAD2/3 signaling, resulting in a block to adipocyte differentiation. (A) After induction of adipogenic differentiation, the KEGG pathway was downregulated in LV-Bmp8b vs LV-ZsGreen1 3T3-L1 cells. (B) The KEGG mapper showed downregulated genes (rectangles highlighted in green) in the PPAR signaling pathway. (C) The PPARγ promoter region is shown in a schematic representation. Three binding sites and sequences of SMAD2/3 TF are predicted. (D) Wild-type and mutation plasmids of predicted SMAD2/3 TF binding sites are shown in a schematic drawing. (E) Quantification the activity of pGL3-Pparγ-promoter and mutation promoter plasmids with pCMV-Bmp8b or vectors in HEK293T. Renilla was used as the internal control. 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 < 0.001).
Fig 2: 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 3: BMP8B triggers NF-κB signaling to suppress adipogenesis. (A) The upregulated KEGG pathway in LV-Bmp8b vs LV-ZsGreen1. (B,C) Western blots and quantification of p-IKKα/β and p-p65 in Mock, LV-ZsGreen1, and LV-Bmp8b. (D,E) Representative photographs of Oil Red O staining were taken to visualize lipids in LV-Bmp8b exposed to JSH-23 with DMSO as a vehicle. The staining intensity was quantified by measuring the optical density at OD492. Scale bar = 20 µm. The symbols in the charts represent three biological replicates. Mean ± SD was used to present the data, which were analyzed using one-way ANOVA (** p < 0.01, *** p < 0.001).
Fig 4: Schematic illustration of BMP8B’s role in modulating adipocyte differentiation. BMP8B binds to the ALK4 to activate SMAD2/3 signaling to suppress the expression of PPARγ to inhibit adipogenesis. Additionally, BMP8B can trigger the NF-κB signal to decrease the activation of PPARγ to inhibit adipogenesis.
Fig 5: BMP8B triggers SMAD2/3 signaling to suppress adipose differentiation via ALK4. (A) Schematic representation of wild-type and GS motif mutants. (B,C) Western blots and quantification of p-SMAD1/5/8 in Mock, LV-ZsGreen1, LV-Bmp8b, and LV-Bmp8b +Alk3-ΔGS cells. (D,E) Western blots and quantification of p-SMAD2/3 in Mock, LV-ZsGreen1, LV-Bmp8b, and LV-Bmp8b + Alk4-ΔGS cells. (F,G) Western blots and quantification of p-SMAD2/3 in Mock, LV-ZsGreen1, LV-Bmp8b, and LV-Bmp8b + Alk5-ΔGS cells. (H,I) Knock down of ALK3, ALK4, and ALK5 in LV-Bmp8b. On Day 8, cells were stained with Oil Red O for quantification. 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 < 0.001).
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