Fig. 2

The Numb and Numbl excisions in osteoblasts inhibit proliferation and differentiation and promote apoptosis. Bone marrow stromal cells (BMSCs) were cultured in complete medium and treated with osteogenic differentiation medium on day 7. Alkaline phosphatase staining (a) was performed on day 7 (red), and Von Kossa staining (b) was performed on day 29 to mark the mineralized matrix (black). c The mRNA levels of osteogenic genes (Alp, Bsp, Ocn and Col1a1) and (d) key transcription factors (Runx2 and Osx) in BMSCs cultures were analysed by RT-qPCR and normalized to β-actin. Data shown represent the mean ± SEM, *P < 0.05. e Western Blot for RUNX2 and OSX. Calvarial osteoblasts isolated from N/NL-floxed mice were transfected with adeno-GFP (CTRL) or adeno-Cre-GFP (ΔN/NL) viruses and cultured in osteogenic medium. GAPDH was used for normalization. f Von Kossa staining was performed 21 days after adenoviral infection. g The qRT-PCR results of bone markers (Alp, Bsp, Ocn, Col1a1, Dmp1 and Phex) two days after Cre transfection. β-actin was used for normalization. Data shown represent the mean ± SEM, *P < 0.05. h Representative images of 5-ethynyl-2’-deoxyuridine (EdU) staining (green) on day 2 to evaluate cell proliferation; nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. i EdU-positive cells were quantified after normalization to DAPI-positive cells. Data shown represent the mean ± SEM, *P < 0.05. j Cell proliferation markers (Cyclin A2, Cyclin D1 and Cyclin E1) in ΔN/NL osteoblasts. β-actin was used for normalization. Data shown represent the mean ± SEM, *P < 0.05. k Dot plots about osteoblast apoptosis resulted from flow cytometry on day 2. Annexin V-PE was scanned in FL2, while 7-AAD was scanned in FL3. l qRT-PCR results for cell apoptosis markers (P53, Bcl-2 and Bcl-x). β-actin was used for normalization. Data shown represent the mean ± SEM, * P < 0.05. m Western Blot for cleaved CASPASE-3. GAPDH was used for normalization