Fig. 3
Knockdown of DEPTOR promotes osteogenic differentiation of hBMSCs in vitro. hBMSCs transfected with lentivirus expressing DEPTOR shRNAs (shDEPTOR #1, shDEPTOR #2) or scrambled control (shNC), and cultured in proliferation medium (PM) or osteogenic medium (OM). a ALP staining on day 7, ARS staining on day 14, and VK staining on day 21 after proliferative culture or osteogenic induction. b, c ALP activity on day 7 and AZR mineralization assay on day 14 after proliferative culture or osteogenic induction. d, e Relative mRNA levels of ALP and RUNX2 measured by qRT-PCR on day 7 of proliferative culture or osteogenic induction. f, g Relative mRNA levels of OSX and OCN measured by qRT-PCR on day 14 of proliferative culture or osteogenic induction. GAPDH used for normalization. h Left: western blot analysis of RUNX2 and OCN protein levels on day 14 after osteogenic induction. GAPDH used as internal control. Right: quantification of band intensities. Data presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, NS not significant (n = 3 independent experiments). See Additional file 3: Figure S2. DEPTOR DEP domain containing mTOR interacting protein, ALP alkaline phosphatase, ARS/AZR Alizarin red S, VK von Kossa, GAPDH glyceraldehyde 3-phosphate dehydrogenase, Ocn osteocalcin, OSX osterix, RUNX2 runt related transcription factor 2