Figure 4: NELL-1 requires integrin β1 to activate Wnt/β-catenin signalling. | Nature Communications

Figure 4: NELL-1 requires integrin β1 to activate Wnt/β-catenin signalling.

From: NELL-1 in the treatment of osteoporotic bone loss

Figure 4

(a–d) RhNELL-1 increases Wnt signalling in the M2-10B4 BMSC line. (a) Active β-catenin immunocytochemistry in M2-10B4 cells, treated with rhNELL-1, WNT3A or control (PBS). (b,c) Western blot analysis and quantification of cytoplasmic and nuclear β-catenin (N=3 wells per treatment). (d) M2-10B4 cells were transfected with TOPFLASH reporter (N=4 wells per treatment). (e,f) RhNELL-1 requires intact Wnt/β-catenin signalling for induction of OB differentiation (N=4 wells per treatment). (e) M2-10B4 cells were treated with PBS or rhNELL-1 with or without DKK-1 for 3 days. Runx2 expression measured using qRT–PCR. (f) M2-10B4 cells were transduced with Runx2-EGFP reporter lentivirus and treated with PBS or rhNELL-1 with or without XAV939. Runx2 reporter assay was performed after 3 days. (g–i) RhNELL-1 increases Wnt signalling in the RAW264.7 osteoclast cell line (N=3 wells per treatment). (g) Gene expression after 2 days with or without rhNELL-1. (h) Active β-catenin immunocytochemistry in RAW264.7 cells, treated with rhNELL-1, WNT3A or control (PBS). (i,j) Western blot and quantification with or without rhNELL-1. (k–p) RhNELL-1 requires integrin β1 to activate Wnt/β-catenin signalling (N=3 wells per treatment). (k,l) siRNA-mediated knockdown of the known NELL-1 receptor integrin β1 was performed in M2-10B4 cells, confirmed by western blot analysis and quantification. (m,n) Similar siRNA-mediated knockdown of integrin β1 was performed in RAW264.7 OC cells. (o,p) After 2 days of rhNELL-1 (300 ng ml−1) treatment, Wnt signalling gene expression was evaluated in either scramble or integrin β1 siRNA-treated M2-10B4 or RAW264.7 cells. Quantitative RT–PCR for CyclinD and Axin2 was performed. Black scale bars, 100 μm. Data points indicate the means, while error bars represent one s.e.m. In vitro experiments were performed in biological triplicate, unless otherwise described. Parametric data were analysed using an appropriate Student’s t-test or a one-way ANOVA, followed by a post hoc Tukey’s test. Nonparametric data were analysed with a Mann–Whitney U-test or a Kruskal–Wallis one-way analysis. *P<0.05, **P<0.01.

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