Fig. 6

Fig. 6 Bmp8a activates Smad2/3 signaling to inhibit adipogenesis through type I receptor ALK4.

a Schematic drawing of wild-type and GS motif mutation (Alk3-ΔGS, Alk4-ΔGS, Alk5-ΔGS) plasmids. b, c Immunoblot analysis and quantification of p-Smad1/5/8 in Mock, LV-ZsGreen1, LV-bmp8a, LV-bmp8a + Alk3-ΔGS, LV-Bmp8a, and LV-Bmp8a + Alk3-ΔGS 3T3-L1 cells (n = 3). d, e Immunoblot analysis and quantification of p-Smad1/5/8 in Mock, LV-ZsGreen1, LV-bmp8a, LV-bmp8a + Alk4-ΔGS, LV-Bmp8a, and LV-Bmp8a + Alk4-ΔGS 3T3-L1 cells (n = 3). f, g Immunoblot analysis of p-Smad1/5/8 in Mock, LV-ZsGreen1, LV-bmp8a, LV-bmp8a + Alk5-ΔGS, LV-Bmp8a, and LV-Bmp8a + Alk5-ΔGS 3T3-L1 cells (n = 3). Protein expression levels were quantified by using ImageJ software and normalized to the amount of total protein. h, i knocked-down ALK3, ALK4, and ALK5 in LV-bmp8a or LV-Bmp8a 3T3-L1 cells, were induced to differentiate. Lipid contents of the resulting adipocyte-like cells were stained and quantified (n = 3). j Schematic diagram of the Pparγ promoter region. Three predicted TF binding sites and sequences. k Schematic drawing of wild-type and predicted TF binding sites mutation plasmids (pGL3-Pparγ-promoter-ΔR1, pGL3-Pparγ-promoter-ΔR2, pGL3-Pparγ-promoter-ΔR3). l Dual-luciferase report assay was used to analyze the abilities of zebrafish bmp8a and mouse Bmp8a in activation of the Pparγ promoter (n = 3). The pGL3-Pparγ-promoter, pGL3-Pparγ -promoter-ΔR1, pGL3-Pparγ-promoter-ΔR2 or pGL3-Pparγ-promoter-ΔR3 was transfected into HEK293T cells along with pCMV-bmp8a, pCMV- Bmp8a or empty vector. After 48 h, the transfected cells were collected for luciferase assays. Renilla luciferase was used as the internal control. Data were from three independent experiments and were analyzed by One-way ANOVA and were presented as mean ± SD (ns not significant, **p < 0.01, ***p < 0.001).