Mi RNA10 b Reciprocally Stimulates Osteogenesis and Inhibits

Mi. RNA-10 b Reciprocally Stimulates Osteogenesis and Inhibits Adipogenesis Partly through the TGF-&#x 003 B 2; /SMAD 2 Signaling Pathway Li Hongling, Fan Junfen, Fan Linyuan, Li Tangping, Yang Yanlei, Xu Haoying, Deng Luchan, Li Jing, Li Tao, Weng Xisheng, Wang Shihua, Chunhua Zhao Robert 1 Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Center of Excellence in Tissue Engineering Chinese Academy of Medical Sciences, Beijing Key Laboratory (No. BZO 381), Beijing 100005, China. 2 Department of Orthopaedic Surgery, Peking Union Medical College Hospital, Peking Union Medical College, Beijing 100730, China. 3 Current address: Department of Orthopaedic Surgery, The Affiliated Hospital of Qingdao University, Qingdao 266003, China. Figure 5. SMAD 2 is a direct target of mi. R-10 b. (A and B) Bioinformatic analysis was performed to predict the binding seed sequence of mi. R-10 b with the 3&#x 02019; UTR of SMAD 2. The wild-type (WT) or mutant-type (MUT) constructs were inserted into the psi. CHECK-2 reporter vector. Luciferase activity was measured in the lysates, and the values were normalized to the psi. CHECK-2 vector. (C) Western blot and q. RT-PCR analyzed the expression of Aging and Disease, 2018, 9(6), 1058 -1073. DOI: 10. 14336/AD. 2018. 0214 SMAD 2 in h. ADSCs. (D) The knockdown efficiency of three SMAD 2 si. RNAs was confirmed by q. RT-PCR and western blot. (E) ALP staining and activity assay were performed to analyze ALP expression and activity on day 6. (F) Alizarin red staining showed increased calcification after SMAD 2 knockdown. (G and H) The critical regulators of osteogenesis were analyzed by q. RT-PCR and western blot. (I) Oil red O staining and extraction were performed to detect the formation of lipid droplets on day 10 of adipogenic differentiation. (J and K) q. RT-PCR and western blot were used to analyze adipogenic-specific markers after SMAD 2 knockdown. The data, normalized to GAPDH, are averages of 3 independent experiments (mean &#x 000 B 1; SD). &#x 0002 A; P &#x 0003 C; 0. 05;