Fig. 3.

PPARδ activates protein and gene expression of perilipin5 in C2C12 cells. A: C2C12 cells were differentiated until day 6 and stimulated 24 h with vehicle (0.1% DMSO) or agonists for PPARα (WY-14643; 10 μM), PPARδ (GW501516; 0.1 μM), or PPARγ (Rosi/BRL-49653, 1 μM; Tro, 1 μM; and GW1929, 1 μM), or an antagonist for PPARγ (GW9662; 1 μM). All wells received equal amounts of vehicle (0.1% DMSO). Relative gene expression of Plin2-Plin5 normalized to 36B4. B: C2C12 cells were transfected with pcDNA3 vector (control) or various pcDNA3-PPAR expression vectors at day 0, differentiated for 3 days and stimulated with selective PPAR activators for 24 h. Activators of PPARα (WY-14643; 10 μM), PPARδ (GW501516; 0.1 μM), or PPARγ (Rosi/BRL-49653; 1 μM). Relative gene expression of Plin2-5 normalized to 36B4. Results are presented as mean ± SD (n = 3, *P > 0.05, **P > 0.01). C: C2C12 cells were transfected with pcDNA3-6xHis-V5-PPARα, -PPARδ, or -PPARγ expression vectors and the relative expression level of each PPAR isoform was determined by Western blot using a His antibody. D, E: C2C12 cells were differentiated in the presence of a PPARδ activator (GW501516; 0.1 μM) and/or BSA-OA (100 μM) from day 0 until day 7. D: Relative gene expression of Plin2-5 normalized to 36B4. Results are presented as mean ± SD (n = 3). Statistical differences refer to BSA (**P < 0.01). E: The short perilipin5 (perilipin5 aa16-463) protein was increased following PPARδ agonist (GW501516) treatment. The Western blot shows two independent samples per treatment. F: C2C12 cells were transfected with pcDNA3-Plin2, -Plin2 aa125-425, -Plin5, or -Plin5 aa16-463 at day 0, differentiated for 3 days, and stimulated with BSA-OA (100 μM) for an additional 24 h. Perilipin2 and perilipin5 protein amounts were analyzed using specific perilipin antibodies. Each lane contains proteins pooled from three independent wells.