Fig. 3

Evaluation of the paracrine/autocrine effect of adiponectin in the mifepristone-induced enhancement of adipocyte differentiation in 3T3-L1 cells. A, B The effect of mifepristone on the secretion of adiponectin during adipogenesis was examined in two protocols. Representative immunoblot (IB) images and summary (n = 4) show the levels of adiponectin secretion with and without mifepristone treatment (0.1 and 1 µM) on days 6, 7 and 8 (A) and days 3, 4 and 5 (B). The adiponectin secretion levels were expressed relative to those obtained without mifepristone on day 6 (A) or day 3 (B). In the schematic illustrations showing the experimental protocols, the dotted lines indicate the period of vehicle treatment, while the solid lines indicate the period of mifepristone treatment. Since bovine adiponectin in FBS interferes with the quantification of secreted mouse adiponectin, the samples were obtained in serum-free medium. C Representative fluorescent microscopic images and summary (n = 4) of Bodipy 493/503 (green) fluorescence on day 12 after the induction of adipogenesis, with and without treatment with 1 µM mifepristone and 12.5 µg/mL adiponectin-neutralizing antibody ANOC 9104 (NAb). Mifepristone and NAb were applied during 5-day treatment with MIX and INS medium. Scale bar, 300 µm. D, E The effects of mifepristone and NAb on the expression of adiponectin and Fabp4 mRNA (D) and adiponectin protein (E) on day 5 after the induction of adipogenesis. The mRNA level was normalized to that of 18S rRNA; the protein level was normalized to that of Rac1. The values obtained with no treatment were assigned a value of 1. F Summary (n = 3) of the effect of 1 µM mifepristone and 10 µM T0070907 on the expression of Fabp4, PPARγ, and PPARγ2 mRNA on day 5 after the induction of adipogenesis according to the protocol shown in Fig. 1A. The levels of Fabp4 and PPARγ were normalized to the level of 18S rRNA, and expressed relative to that obtained without any treatment. All data represent the mean ± S.E.M. *P < 0.05; **P < 0.01 vs. day 6 (A), day 3 (B) or as indicated (C–F). ^†P < 0.05; ^††P < 0.01 vs. vehicle (A and B)