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. 2011 Jun 28;36(5):725–734. doi: 10.1038/ijo.2011.124

Figure 4.

Figure 4

Effects of CM from cells stably overexpressing BMP-3b on adipogenesis. (a) CM from BMP-3b-overexpressing or wild-type CHO cells (control) was subjected to western blot analysis with an anti-BMP-3b antibody. The arrow indicates the BMP-3b protein. CM aliquots (10 μl) were subjected to western blot analysis. CM1, medium with 10% FBS; CM2, medium without FBS; CM3, 10-fold concentrated CM2. (b) Quantitative RT-PCR analysis of adiponectin on day 8 after 3T3-L1 cells were treated with each CM described in panel a. The expression levels were normalized to S18 levels. Relative gene expression is shown as the ratio compared with the control. (c) Quantitative RT-PCR analysis of adipogenic markers during adipocyte differentiation of 3T3-L1 cells that were treated with CM1. The expression levels were normalized to S18 levels. Relative gene expression is shown as the ratio compared with the expression levels of the control at day 0. (d) CM1 from panel a was subjected to a 3bpro antibody IgG immunoaffinity gel column, and the pass-through fraction was analyzed by western blotting with an anti-BMP-3b antibody. The arrow indicates the BMP-3b protein. CM aliquots (10 μl) were subjected to western blot analysis. −, CM1 before 3bpro antibody IgG immunoaffinity gel column treatment; +, the CM1 pass-through fraction of the column. (e) Oil Red O staining of differentiated 3T3-L1 cells that were incubated with CM from panel d for 8 days. (f) Quantitative RT-PCR analysis of adipogenic markers using the cells described in panel e. The expression levels were normalized to S18 levels. The relative gene expression is shown as the ratio compared with the control. (b, c, f) Data are represented as the means±s.e.m. (n=3). *P<0.05 versus control. **P<0.01 versus control; #P<0.01 versus BMP-3b CM1 or CM3. Experiments were performed three times with independent cultures.