Fig. 3

Suppressive activity of PPE separated by ultrafiltration and gel-filtration chromatography for adipogenesis. a 3T3-L1 cells were cultured under differentiation conditions with or without each ultrafiltrated fraction, < 3 kDa or > 3 kDa separated from PPE using an Amicon. After 8 days of differentiation culture, the cells were stained with Oil Red O, and lipid droplets in the cells were imaged by bright field microscopy. Each fraction was diluted with the culture medium at 1:100. b Stained lipid droplets were extracted with isopropanol and quantified by measuring the absorbance at 492 nm. c PPE was fractionated by gel-filtration chromatography on a Superdex peptide 10/300 GL column. The fraction numbers are shown on the chromatogram (fractions No. 4–10). d 3T3-L1 cells were cultured under a differentiation condition with or without each fraction (fractions No. 4–10) until day 8. After the cells were stained with Oil Red O, lipid droplets in the cells were imaged by bright field microscopy. e Stained lipid droplets were extracted with isopropanol and quantified by measuring the absorbance at 492 nm. f The gene expressions of adipocyte differentiation markers (Cebpa, Pparg and Adipoq) in 3T3-L1 cells cultured with fraction No. 9 under a differentiation condition were analyzed on day 8 by RT-qPCR. Control represents cells cultured without fraction No. 9, and the 3T3-L1 represents cells cultured without either fraction No. 9 or differentiation-inducing agents. Vehicle represents the cells cultured with PBS instead of each fraction. The gene expressions in 3T3-L1 cells cultured with fraction No. 9 are presented relative to the value in 3T3-L1. Experiments were performed in triplicate, and the data are presented as the mean ± SEM (n = 3). *p < 0.05, ***p < 0.005 vs. Control. Experiments were repeated at least three times, and representative results are shown