Figure 2.
3T3-L1 cells were treated with DM or ciglitazone (15 μM) to initiate differentiation and were evaluated on days 8 or 10. (a) COX inhibition fails to modulate DM-induced adipocyte differentiation. The extent of TAG formation was estimated in cells differentiated with DM, ciglitazone (Cig), or DM in combination with indomethacin (3 μM) or NS-398 (10 μM). (b) PG formation is not enhanced during adipogenesis. Products during adipogenesis, as determined by Northern blotting (PPARγ, aP2, GAPDH), RT-PCR (COX-1), ribonuclease protection assay (COX-2) analysis, and LC-MS-MS (PGE2, 15d-PGJ2) in the medium (med) and cells. (c) Inhibition of PG formation by indomethacin fails to modulate the induction of adipocyte protein expression by DM. (d) Inhibition of PG formation fails to influence accumulation of lipid induced by DM. Oil red O staining of cells illustrates lipid accumulation in untreated cells versus cells treated with DM ± indomethacin. (e) 15d-PGJ2 drives adipogenesis in a PPARγ-dependent manner. Cells were treated with 15d-PGJ2 (0.1–15 μM) for 2 days in the absence (open circle) or presence (filled circle) of the PPARγ antagonist BADGE (100 μM). (f) Low concentrations of 15d-PGJ2 do not amplify the effect of ciglitazone. Cells were differentiated with ciglitazone alone (filled circle) or in combination with 1 μM (open circle) or 0.1 μM 15d-PGJ2 (filled triangle). (g) COX inhibition fails to modulate the enhancement of ciglitazone-dependent differentiation by DM. Cells were incubated to differentiate by adding ciglitazone alone (filled triangle) or in combination with DM (filled circle) or DM + indomethacin (open circle).