Table 1.
Primers for PCR.
Figure 1.
PGE2-induces adipogenic differentiation in rat TSCs.
Rat TSCs were incubated with 0, 10, 50, 100, or 200/ml PGE2 for 7 days. (A) Adipogenic differentiation detected by Oil Red O staining; (B) the Oil Red O staining areas in the cells were assessed as described in the text; (C) PPARγ mRNA levels determined by qRT-PCR. Rat TSCs were incubated with 100 ng/ml PGE2 for 0, 3, 7, or 10 days. (D) Adipogenic differentiation detected by Oil Red O staining; (E) the areas stained with Oil Red O in the cells were assessed; (F) PPARγ mRNA levels determined by qRT-PCR. The mRNA levels were normalized using GAPDH. Results represent the mean ± SD. *P<0.05, **P<0.01 with respect to TSCs without PGE2.
Figure 2.
BMP-2 alone does not induce adipogenic differentiation in rat TSCs.
TSCs were incubated with (0, 10, 50, 100, or 200 ng/ml BMP-2 for 7 days. (A) Adipogenic differentiation detected by Oil Red O staining; (B) the areas stained with Oil Red O in the cells were assessed; (C) PPARγ mRNA levels determined by qRT-PCR. TSCs were incubated with 100 ng/ml BMP-2 for 0, 3, 7, or 10 days. (D) Adipogenic differentiation detected by Oil Red O staining; (E) the Oil Red O staining areas in the cells were assessed; (F) PPARγ mRNA levels determined by qRT-PCR. The mRNA levels were normalized using GAPDH. Results represent the mean ± SD.
Figure 3.
IGF-1 expression in rat TSCs is upregulated by PGE2 via the cAMP/PKA/CEBPδ pathway.
TSCs were incubated with 0, 10, 50, 100, or 200/ml PGE2 for 7 days. (A) IGF-1 mRNA expression was determined by qRT-PCR; (B) IGF-1 protein levels were quantitated by ELISA. TSCs were incubated with 100 ng/ml PGE2 for 0, 3, 7, or 10 days. (C) IGF-1 mRNA expression was determined by qRT-PCR; (D) IGF-1 protein levels were quantitated by ELISA. (E) TSCs were incubated in medium containing PGE2 (100 ng/ml), with or without the cAMP inhibitor ddA (10 μmol/l) and intracellular cAMP levels were determined by EIA. TSCs were incubated in medium containing PGE2 (100 ng/ml) with or without the cAMP inhibitor ddA (10 μmol/l), the PKA inhibitor H-89 (10 μM), or after transduction with the CEBPδ shRNA. (F) p-PKA and total PKA, nuclear (nuc) and cytoplasmic (cyto) CEBPδ protein levels determined by western blotting. HDAC (nuc) and β-actin (cyto) were used as loading controls; (G) IGF-1 mRNA levels determined by qRT-PCR. The mRNA levels were normalized using GAPDH; (H) IGF-1 protein levels determined by ELISA. Results represent the mean ± SD. *P<0.05, **P<0.01 with respect to TSCs without PGE2; #P<0.05 with respect to TSCs with PGE2.
Figure 4.
IGF-1 and BMP-2 induce adipogenic differentiation of rat TSCs.
TSCs were incubated with 0, 1, 5, 10, or 20-1 and 100 ng/ml BMP-2 for 7 days. (A) Adipogenic differentiation detected by Oil Red O staining; (B) the Oil Red O staining areas in the cells were assessed; (C) PPARγ mRNA levels determined by qRT-PCR. TSCs were incubated with 10 nM IGF-1 plus 100 ng/ml BMP-2 for 0, 3, 7, or 10 days. (D) Adipogenic differentiation detected by Oil Red O staining; (E) the Oil Red O staining areas in the cells were assessed; (F) PPARγ mRNA levels determined by qRT-PCR. The mRNA levels were normalized using GAPDH. Results represent the mean±SD. *P<0.05, **P<0.01 with respect to TSCs without PGE2.
Figure 5.
Knock down endogenous IGF-1 and BMP2 abolished PGE2-induced adipogenic differentiation.
Rat TSCs were treated with PGE2 (100 ng/ml) with or without BMP-2 shRNA or IGF-1 shRNA. (A) Levels of IGF-1 and BMP2 were determined by western blotting. β-actin was used as loading controls. (B) Adipogenic differentiation detected by Oil Red O staining; (C) The Oil Red O staining areas in the cells were assessed; (D) PPARγ mRNA levels determined by qRT-PCR. The mRNA levels were normalized using GAPDH. Results represent the mean ± SD. **P<0.01 with respect to TSCs without PGE2; #P<0.05 with respect to TSCs with PGE2.
Figure 6.
CREB and Smad are phosphorylated by IGF-1 and BMP-2, respectively, in PGE2-induced adipogenic differentiation.
Rat TSCs were treated with PGE2 (100 ng/ml), IGF-1 (10 nM), BMP-2 (100 ng/ml), or IGF-1 (10 nM) + BMP-2 (100 ng/ml) with or without CREB shRNA or Smad shRNA. (A) Levels of total and phosphorylated CREB and Smad were determined by western blotting. β-actin was used as loading controls. TSCs were incubated with PGE2 (100 ng/ml), or IGF-1 (10 nM) + BMP-2 (100 ng/ml), with or without CREB shRNA or Smad shRNA. (B) Adipogenic differentiation detected by Oil Red O staining; (C) the Oil Red O staining areas in the cells were assessed; (D) PPARγ2 mRNA levels determined by qRT-PCR. The mRNA levels were normalized using GAPDH. Results represent the mean ± SD. **P<0.01 with respect to TSCs without PGE2; #P<0.05 with respect to TSCs with PGE2.