Fig 2: 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.

Fig 3: BMP-3b is secreted from 3T3-L1 cells as a non-covalent complex. (a) Conditioned media from CHO and 3T3-L1 cells that were transiently transfected with a Flag-tagged BMP-3b expression plasmid were immunoprecipitated with an anti-Flag antibody and then subjected to western blot analysis with the 3bpro antibody. IP, immunoprecipitation antibody; IB, immunoblotting antibody; mature, antibody specific for the mature region; pro, antibody specific for the pro-region. The deduced structures and molecular weights (kDa) are shown. The precursor form consists of the pro- (open box) and the mature (closed box) regions. Mock, vector control; pre, preadipocytes; adipo, mature adipocytes. The asterisk indicates a non-specific band. The samples were electrophoresed under reducing conditions. Note that the anti-Flag antibody recognizes the mature region. (b) CM from 3T3-L1 cells that were transfected with a BMP-3b expression plasmid was immunoprecipitated with a 3bpro antibody and then subjected to western blot analysis with a 3bpro antibody or an anti-BMP-3b mature antibody. (c) CM from 3T3-L1 cells was immunoprecipitated with a 3bpro antibody and then subjected to western blot analysis with an anti-BMP-3b mature antibody. (d) The biosynthetic pathway of BMP-3b in 3T3-L1 cells. The representative molecular forms and potential proteolytic processing sites (triangle) are illustrated schematically. The data are representative of at least three independent experiments.

Fig 4: The molecular form of BMP-3b that is secreted from 3T3-L1 cells. (a) Conditioned media from 3T3-L1 and CHO cells that were transiently transfected with a Flag-tagged BMP-3b expression plasmid were immunoprecipitated with an anti-Flag antibody and then subjected to western blot analysis with an anti-Flag antibody. IP, immunoprecipitation antibody; IB, immunoblotting antibody; mature, antibody specific to the mature region. The deduced structures and molecular weights (kDa) are shown. The precursor form consists of the pro- (open box) and the mature (closed box) regions. Mock, vector control; pre, preadipocytes; adipo, mature adipocytes. The asterisk indicates a non-specific band. Note that the anti-Flag antibody recognizes the mature region. (b) Diagram representing the BMP-3b precursor. The site (•••) used for antibody (3bpro) production is shown. The potential proteolytic processing sites are indicated by triangles. (c) CM from CHO and 3T3-L1 cells that were transfected with a BMP-3b plasmid was purified with Heparin–Sepharose and then subjected to western blot analysis with the 3bpro antibody. pro, antibody for the pro-region. (a, c) Samples were electrophoresed under reducing conditions and the results are representative of at least three independent experiments. The positions of the molecular mass markers are shown on the left (a) or in the middle (c) of the panels.

Fig 5: Expression of BMP-3b and other BMPs in 3T3-L1 cells. (a, c) RT-PCR analysis of BMP-3b, other BMPs and adipogenic markers during the differentiation of 3T3-L1 cells. The products were separated on 2% agarose gels and visualized with ethidium bromide. The positive control (P) is RNA from calvariae for BMP-3b, BMP-3 and BMP-2; lung for BMP-4 and BMP-6; and brain for BMP-7. N, negative control (H2O). (b) Quantitative RT-PCR analysis of BMP-3b in 3T3-L1 cells. The data are presented as the means±s.e.m. of three independent experiments (n=3). a.u., expression levels normalized to 18S levels. *P<0.05 versus preadipocytes. pre, preadipocytes (day 0); adipo, mature adipocytes (day 8).