Fig. 6

Effects of PPE on IDM-induced phosphorylation of MAPK. (A and B) 3T3-L1 cells which reached confluence were cultured with IDM in the presence of fraction No. 9 (Fraction No. 9/IDM 1 h) or PBS (Vehicle/IDM 1 h) for 24 h (a) or 0–48 h (b). The cell lysates were subjected to western blotting analysis with antibodies against phospho-p38 MAPK, total p38 MAPK, phospho-ERK1/2, total ERK1/2 or β-Actin. The relative intensity of each band of the phosphorylated forms after normalization for the levels in the total forms and β-Actin is shown as a bar graph. The experiment in (a) was performed in triplicate, and the data are presented as the mean ± SEM (n = 3). ***p < 0.005 vs. Vehicle/IDM 1 h. c 3T3-L1 cells which reached confluence were cultured with IDM for 2 days. The medium was changed every 48 h to fresh medium containing insulin until day 8. Fraction No. 9 was added during days 0–2, and 10 μM SB208035 or DMSO was also added during days 0–2 with 1 h of pre-treatment. After the cells were stained with Oil Red O, lipid droplets in the cells were imaged with a bright field microscope. d The gene expressions of adipocyte differentiation markers (Cebpa, Pparg and Adipoq) were analyzed on day 8 by RT-qPCR. Control represents cells cultured without fraction No. 9, and Fraction No. 9 represents cells cultured with fraction No. 9. Fraction No. 9/DMSO and Fraction No. 9/SB203580 represent cells cultured with fraction No. 9 in the presence of DMSO or 10 μM SB203580, respectively. The gene expression levels are presented as a ratio of the value in Control cells. Experiments were performed in triplicate, and the data are presented as the mean ± SEM (n = 3). ***p < 0.005 vs. Control