Fig. 1. Effects of genistein on adipocyte differentiation in 3T3-L1 cells. Confluent 3T3-L1 cells were exposed to differentiation cocktail (1 μM dexamethasone, 5 μg/ml insulin, and 0.5 mM IBMX) for 2 days, and cells were changed into normal DMEM in the presence or absence of genistein for the indicated concentrations (A) or cells exposed to genistein for the indicated time period (B). These cells were fixed with 3.5% formaldehyde in PBS and fat drops were stained by Oil-Red O dye staining.

Fig. 2. Genistein also induced apoptosis of mature 3T3-L1 cells. Cells were fully differentiated at day 8 and exposed to genistein for indicated concentrations. After inducing apoptosis by genistein, apoptosis was detected either by MTT assay or Hoechst33342 dye.

Fig. 3. The effects of AMPK activation by genistein. 3T3-L1 cells were fully differentiated by differentiation cocktail, cells were exposed to genistein (100 μM) for the indicated time periods (A) or various concentrations, respectively, (B) and AMPK activation and its substrate acetyl-CoA carboxylase (ACC) phosphorylation were detected by Western blot analysis. Also differentiated cells were exposed to genistein for 12 h in the presence or absence of NAC (5 mM). After additional incubation for 30 min in the presence of 10 μM DCFH-DA the changes in fluorescence intensity were measured by fluorescence-activated cell scanning analysis. Under the same conditions, the phosphorylation level of ACC-Ser⁷⁹ (P-ACC) were examined (C).

Fig. 4. Effects of AICAR on adipocyte differentiation and apoptosis of mature adipocytes. Confluent 3T3-L1 cells were exposed to differentiation cocktail (1 μM dexamethasone, 5 μg/ml insulin, and 0.5 mM IBMX) for 2 days, and cells were changed into normal DMEM in the presence or absence of AICAR for the indicated concentrations (A). These cells were fixed with 3.5% formaldehyde in PBS and fat drops were stained by Oil-Red O dye staining or apoptosis was detected with Hoechst33342 dye.

Fig. 5. Similar effects of capsaicin and EGCG on AMPK activation and adipocyte differentiation interference. 3T3-L1 pre-adipocyte were pretreated with EGCG or capsaicin (100 μM) for 30 min and rapidly incubated with differentiation medium. After each time period, AMPK activation and adipocyte differentiation were detected with either Western blot analysis or Oil-Red O staining.