To induce adipocyte differentiation by BMPs in the absence of induction cocktails, both WT brown preadipocytes and 3T3-L1 white preadipocytes were grown in regular growth medium supplemented with combination of rhBMPs (3.3 to 8.3 nM), insulin (20 nM) and T3 (1 nM) or vehicle as indicated in the text and figure legends for 7–13 days. To stimulate thermogenic program, differentiated cells were incubated with 500 µM dibutyrul cyclic AMP for 4 hrs. Cells were grown in growth medium without hormonal supplements for 18 hrs prior to cAMP stimulation.
C3H10T1/2 cells were grown in the presence and absence of 8.3 nM rhBMP-7 for 3 days to reach confluence (day 3). These cells were then induced to adipocyte differentiation using protocols described below for additional 7 days (day 10). Adipocyte differentiation was done by treating confluent cells for 48 hours in medium supplemented with 20 nM insulin and 1 nM triiodothyronine (T3), 0.5 mM isobutylmethylxanthine (IBMX), 5 µM dexamethasone, and 0.125 mM indomethacin. Cells were placed back to growth medium supplemented with insulin and T3, which was then changed every second day. After four to five more days in this medium, cells exhibited a fully differentiated phenotype with massive lipid accumulation.
C3H10T1/2 cells were grown in the presence and absence of 8.3 nM rhBMP-7 for 3 days to reach confluence (day 3). These cells were then induced to adipocyte differentiation using protocols described below for additional 7 days (day 10). Adipocyte differentiation was done by treating confluent cells for 48 hours in medium supplemented with 20 nM insulin and 1 nM triiodothyronine (T3), 0.5 mM isobutylmethylxanthine (IBMX), 5 µM dexamethasone, and 0.125 mM indomethacin. Cells were placed back to growth medium supplemented with insulin and T3, which was then changed every second day. After four to five more days in this medium, cells exhibited a fully differentiated phenotype with massive lipid accumulation.
