Fig. 3
From: NUMB maintains bone mass by promoting degradation of PTEN and GLI1 via ubiquitination in osteoblasts
The Numb and Numbl deficiency promoted PTEN instead of Notch. a Triplicate qRT-PCRs to measure the expression of Notch target genes (Hes1, Hes5, Hey1 and HeyL). b Western Blots for NUMB, NICD, HES1 and PTEN changes in ΔN/NL osteoblasts. Whole-cell lysates and RNA were extracted two days after adenovirus transduction. GAPDH was used for normalization. c ΔN/NL and CTRL osteoblasts were transiently transfected with an Rbp-jk reporter or positive/negative control plasmids. Extra controls of Ad-NICD (positive) and DAPT (negative) were set up to observe the efficiency of this test. Luciferase levels were normalized to Renilla luciferase and presented as fold changes to monitor Notch activation. Error bars represent the standard deviation of triplicate transfections. Data shown represent the mean ± SEM, *P < 0.05. d Two images of femoral paraffin sections of WT and DKO mice immunostained by anti-PTEN (red) and counterstained with haematoxylin (blue nucleus). Black and blue arrows refer to positive signals on or near the bone surface, respectively. Scale bar, 100 μm. e A PTEN-specific inhibitor rescued depressed osteogenesis in ΔN/NL osteoblasts. VO-Ohpic (5 µM) was incubated with ΔN/NL osteoblasts. Alizarin Red was used to mark the mineralized matrix here. f Bone markers (Ocn, Col1a1 and Dmp1); proliferation markers (Cyclin A2, Cyclin D1) and apoptosis markers (P53, Bcl-2). Data were normalized to β-actin. Data shown represent the mean ± SEM, *P < 0.05. Western blots for signal molecules in the Akt (g) and mTOR pathways (h). GAPDH was used for normalization
