Fig. 6

Knockdown of DEPTOR activates BMP4 signaling through MEG3. a Relative mRNA expression of BMP4 in MEG3 knockdown (si-MEG3 #1, si-MEG3 #2) and control vector (si-NC) groups. GAPDH used for normalization. b Western blot analysis of BMP4 and its downstream Smad expression in MEG3 knockdown and NC groups. GAPDH utilized as internal control. c, d MEG3 siRNA (si-MEG3) introduced into DEPTOR knockdown hBMSCs. c Relative mRNA expression of BMP4 measured using qRT-PCR. GAPDH used for normalization. d Western blot analysis of BMP4 and its downstream Smad expression. GAPDH utilized as internal control. e MEG3 promoter and location of primers. Positions marked relative to the transcription start site (TSS). f ChIP-qPCR showing interaction between DEPTOR and MEG3 promoter in hBMSCs. IgG used for normalization. Data presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, NS not significant (n = 3 independent experiments). g Proposed mechanism of DEPTOR modulation of osteogenesis via regulation MEG3-mediated BMP4 activation. BMP4 bone morphogenetic protein 4, Chip chromatin immunoprecipitation, DEPTOR DEP domain containing mTOR interacting protein, GAPDH glyceraldehyde 3-phosphate dehydrogenase, MEG3 maternally expressed 3 (nonprotein coding), shNC scrambled control, sh short hairpin, si short interfering