Figure 3
15d-PGJ2 inhibits VCAM-1 in PPARγ independent manner. bEND.3 cells were treated with GW9662 (10 μM) 30 min prior to treatment with 15d-PGJ2 (10 μM) or troglitazone (10 μM) followed by TNFα treatment (50 ng/ml). bEND.3 cells were lysed and processed for immunoblot analysis for VCAM-1 and β actin expression (A). Endothelial cell line was cotransfected with PPARγ-GAL4 chimeras and the reporter plasmid (upstream activating sequences)5-TK-CAT. After 48 h, cells were treated with 15d-PGJ2 or trogliatzone in the presence or absence of GW9662 (10 μM) for 24 h. Cell extracts were subsequently assayed for CAT activity by ELISA (Roche) (B). pCMV-GAL4-binding domain (without insert) and (upstream activating sequences)5-TK-CAT were transfected as a control to detect the basal levels of CAT activity (first lane). Data are mean of three values ± SD. *** p < 0.001 as compared with untreated cells; !!! p < 0.001 as compared with 15d-PGJ2 treated cells. (C) Cells were transfected with PPARγ wild type (Wt) and dominant negative (DN) constructs followed by treatment with 15-dPGJ2 (5 and 10 μM; 30 min) and TNFα (50 ng/ml, 2 h) and processed for qPCR for detection of VCAM1 mRNA expression as described in 'Material and Methods' (C). Results were calculated as mean ± SD for 3 independent experiments. Samples were run in triplicates. &&& p < 0.001 compared with control (untreated and unstimulated cells) and !!! p < 0.001 as compared to TNFα treatment. Cells were co-transfected with PPARγ wild type (Wt) and dominant negative (DN) (0.5 μg/well) constructs along with PPRE-luc reporter (0.5 μg/well) and pRL-TK (0.5 μg/well) followed by treatment with 15-dPGJ2 (10 μM) after 24 h. After 24 h incubation, luciferase activity was performed, as described before pcDNA3.1 was added to normalize the total content of DNA for transfection. Data are mean ± SD of three different values. ***, p < 0.001 as compared with untreated cells; !!!, p < 0.001 as compared with 15d-PGJ2-treated PPAR wt transfected cells.