The mouse 3T3-L1 pre-adipocyte cell line was obtained from the American Type Culture Collection (Manassas, VA, USA) and was maintained at 37°C and 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM; GE Healthcare Life Sciences, Chalfont, UK) supplemented with 10% calf serum (CS) or 10% fetal bovine serum (FBS; Zhejiang Tianhang Biotechnology Co., Ltd., Huzhou, China), as indicated for each procedure, plus 100 U/ml penicillin, and 100 mg/ml streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Myr, SFN, insulin (INS), 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (DEX), and Oil Red O dye were purchased from Merck KGaA. Primary antibodies against Akt (catalog no. 9272), phosphorylated (p)-Akt at Ser473 (p-Akt^Ser473; catalog no. 4060), ribosomal protein S6 kinase β-1 (alternatively known as p70S6K1; catalog no. 2708), p-p70S6K1 (catalog no. 9208), caspase 3 (catalog no. 9662), poly-ADP-ribose-polymerase (PARP; catalog no. 9532), the B-cell lymphoma-2 (Bcl-2; catalog no. 3498) apoptosis regulator, Bcl-2-associated death promoter (Bad; catalog no. 9292), and p-Bad at Ser112 (p-Bad^ser112; catalog no. 9291) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Primary antibodies against Bcl-2 associated X (Bax) apoptosis regulator (catalog no. AB026) was purchased from Beyotime Institute of Biotechnology (Haimen, China). The primary antibody against β-actin was purchased from Santa Cruz Biotechnology, Inc., Dallas, TX, USA (catalog no. sc-130656). The horseradish peroxidase-conjugated secondary antibodies (catalog no. BM 2006 and BA1025) were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

3T3-L1 pre-adipocytes cultured until ~90% confluent (D₀) following 2 days of incubation with 10% CS/DMEM growth medium. The medium was then replaced with 10% FBS/DMEM supplemented with 10 µg/ml INS, 1 µM DEX and 0.5 mM IBMX to induce differentiation. Following 2 days (D₂), the medium was replaced with 10% FBS/DMEM containing 10 µg/ml INS for a further 2 days (D₄). This was followed by culture in the 10% CS/DMEM growth medium for an additional 6 days; during which time the medium was refreshed every other day. At 10 days following induction of differentiation (D₁₀), >90% of the cells consisted of mature adipocytes, which contained an abundance of lipid droplets and were ready for treatment.

At the end of differentiation (D₁₀), the mature adipocytes were washed with phosphate-buffered saline (PBS) and fixed with 10% neutral buffered formalin for 30 min at room temperature. The cells were then washed with PBS and subsequently stained with filtered 0.5% (w/v) Oil red O solution for 30 min at room temperature in the dark. Cells were then washed three times with distilled water. Photographs of the Oil Red O-stained cells were captured using a Nikon digital camera system (Nikon Corporation, Tokyo, Japan). A total of 99% (v/v) isopropanol was then added to the cells and incubated for 5 min at room temperature to remove the dye, and the lipid content of the cells was quantified by spectrophotometry at 570 nm.

The viability of mature adipocytes was determined using an MTT assay kit (Beyotime Institute of Biotechnology, Haimen, China), which measures the production of formazan by catalytically active cells and therefore provides a measure of the number of viable cells. Pre-adipocytes were seeded onto a 96-well plate at a density of 2.5×10³ cells/well and induced to differentiate into mature adipocytes using the aforementioned methods. At D₁₀ the mature adipocytes were treated with S40 and/or M100 for 24 h. Following treatment, the cells were incubated with 0.5 mg/ml MTT reagent for 3 h at 37°C to allow the formation of formazan crystals. Dimethyl sulfoxide (150 µl) was subsequently added to each well to dissolve the formazan crystals. The optical density (OD) was determined by measuring the absorbance of wells at 570 nm using a microplate reader. Inhibition of cell viability was calculated as follows: Inhibition (%) = [1-(OD_{treated adipocytes} /OD_{untreated adipocytes})] ×100.

Pre-adipocytes were seeded in 6-cm culture dishes (4×10⁴ cells/dish) coated with poly-lysine, and induced to differentiate into mature adipocytes using the aforementioned methods. Mature adipocytes were then treated with S40 and/or M100 for 24 h. The cells were subsequently fixed with 4% paraformaldehyde at room temperature for 10 min, washed twice with PBS and stained with 10 µg/ml Hoechst 33258 solution for 5 min in the dark. Cells were washed with cold PBS and a drop of anti-fade mounting medium (Beyotime Institute of Biotechnology) was added. Alterations in nuclear morphology were observed under an inverted fluorescence microscope (Olympus IX51 Inverted Microscope; Olympus Corporation, Tokyo, Japan).

The ApoStrand™ ELISA Apoptosis Detection kit (Enzo Life Sciences, Inc., Farmingdale, NY, USA) was used to quantify the levels of single-stranded DNA (ssDNA) generated during apoptosis, according to the manufacturer's instructions. Briefly, mature 3T3-L1 adipocytes (2.5×10³ cells/well) were transferred to 96-well plates following treatment with S40 and/or M100 for 24 h. The cells were fixed at room temperature for 30 min with 4% formamide, and then the ssDNA in apoptotic cells was stained with a mixture of the primary antibody and the peroxidase-labeled secondary antibody provided with the kit. The cells were subsequently incubated with peroxidase substrate at room temperature for 45 min, and the absorbance at 405 nm was read.

Following treatment with S40 and/or M100 for 24 h, ~4×10⁷ mature adipocytes were harvested for extraction of mitochondrial and cytoplasmic protein, and ~1×10⁷ cells were used to extract total protein. Briefly, 2 ml mitochondrial separation reagent containing 1 mM PMSF (Beyotime Institute of Biotechnology) was added, and then shaken at 4°C for 3 min followed by centrifugation at 600 × g for 10 min at 4°C for 10 min. The supernatant (cytoplasm) and the precipitation (mitochondria) were separated and lysed in Nonidet P-40 lysis buffer on ice for 1 h. The supernatant was collected by centrifugation at 12,000 × g for 15 min at 4°C. The protein concentration was quantified using an enhanced bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Protein samples (50 µg/lane) were electrophoresed by 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane. The membranes were blocked in blocking buffer containing 5% nonfat dry milk in Tris-buffered saline with 0.05% Tween 20 (TBST) for 1 h at room temperature. The membranes were then probed with the specific primary antibodies (diluted 1:1,000) overnight at 4°C in TBST. The membranes were subsequently washed with TBST three times for 5 min each time, and incubated with the horseradish peroxidase-conjugated secondary antibodies (diluted 1:10,000) for 1.5 h at room temperature. Membranes were washed again with TBST, developed with an enhanced chemiluminescence kit (Roche Applied Science, Penzberg, Germany) and exposed to X-ray film in a dark room. The protein bands were analyzed using a Gel Doc™ XR⁺ imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Data are expressed as the mean ± standard deviation of at least three independent experiments. SPSS version 10.0 (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. One-way analysis of variance and the Student-Newman-Keuls post hoc test were used to evaluate statistical differences among groups. P<0.05 was considered to indicate a statistically significant difference.

At D₁₀ of adipocyte differentiation, the vast majority of 3T3-L1 cells appeared to contain Oil Red O-stained lipid droplets, which indicated that adipocyte maturation had occurred (Fig. 1).

The results of the MTT assay revealed that treatment with S40 or M100 reduced the viability of mature adipocytes by 14.74±0.02 and 9.36±0.03%, respectively, when compared with untreated cells (P<0.05; Fig. 2A, Table I). However, when S40 and M100 were applied in combination, cell viability was reduced by 33.07±0.02% when compared with the untreated cells (P<0.05; Table I). Treatment with these agents together did not demonstrate an additive effect, as this would have led to a decrease in cell viability of 24.10±0.05% (P<0.05; Table I), which suggests that the observed effect was synergistic.

Oil Red O staining revealed that treatment with SFN and/or Myr decreased the number of lipid droplets that accumulated in mature adipocytes (Fig. 2B). Quantitative spectrophotometry analysis revealed that S40 and/or M100 treatment significantly reduced the lipid content of the cells when compared with untreated cells (P<0.05; Fig. 2C, Table I). Combined treatment with S40 and M100 was not associated with a significant reduction in cellular lipid content when compared with S40 or M100 treatment alone (Table I).

Hoechst 33258 nuclear staining of mature adipocytes revealed that cells displayed characteristic features of apoptosis, including shrinkage, nuclear fragmentation, and chromatin condensation following treatment with SFN and/or Myr for 24 h (Fig. 3A). Apoptosis was further assessed by quantifying the level of ssDNA in cells, which may be used as a marker of apoptotic cells. The ssDNA levels indicated that, while SFN and Myr treatment alone induced apoptosis in adipocytes when compared with the untreated control cells, the effect of combined treatment was significantly greater than that of each compound alone (P<0.05; Fig. 3B). Compared with the control, S40 and M100 increased the levels of apoptosis by 27.38±0.08 and 9.74±0.08%, respectively (P<0.05; Table I). However, combined treatment with S40 and M100 increased apoptosis by 108.04±0.18% when compared with the control (P<0.05; Table I). The calculated additive effect would have led to an increase in apoptosis levels of 37.11±0.16% when compared with the controls (P<0.05; Table I), which therefore suggests that SFN and Myr act synergistically to induce apoptosis in adipocytes.

Bcl-2 family proteins are critical regulators of the mitochondrial apoptotic pathway. The ratio of Bax to Bcl-2 is important in determining cell fate (25). In the present study, SFN treatment markedly increased Bax protein expression levels, while Myr demonstrated no observable effect when compared with the control (Fig. 4A). Neither SFN nor Myr treatment demonstrated any observable effect on Bcl-2 expression levels at the concentrations tested (Fig. 4A). However, treatment with SFN and Myr combined increased Bax expression and decreased Bcl-2 expression levels when compared with the control, thus altering the Bax/Bcl-2 ratio in favor of apoptosis (Fig. 4A). In addition, combined treatment markedly increased Bad expression in the mitochondria when compared with the control, which suggests that Bad translocation from the cytoplasm to the mitochondria was activated, thus promoting apoptosis (Fig. 4B). Furthermore, phosphorylation of Bad at Ser112, which is thought to be necessary and sufficient to promote cell survival, was markedly decreased in the cells treated with SFN plus Myr compared with control cells or cells treated with each compound alone (Fig. 4B). Caspase 3 is the main executor caspase of the majority of programmed cell-death processes (27). As demonstrated in Fig. 4A, treatment with either SFN or Myr for 24 h led to an increase in the levels of cleaved caspase 3 and increased cleavage of the downstream DNA-repair enzyme, PARP, from its native 116 kDa form to the inactive 89 kDa form. As expected, combined treatment additionally induced caspase 3 cleavage and PARP inactivation (Fig. 4A).

Western blot analysis revealed that SFN or Myr treatment alone did not demonstrate any observable effect on Akt phosphorylation at Ser473; however, combined treatment with these agents markedly inhibited Akt phosphorylation when compared with the controls (Fig. 4C). Total Akt expression levels were unaffected in cells treated with SFN and/or Myr (Fig. 4C). As expected, combined SFN and Myr treatment was associated with an observable decrease in the levels of p-p70S6K1 when compared with controls (Fig. 4C). SFN treatment alone decreased p70S6K1 phosphorylation; however, this effect was not as pronounced as combined SFN and Myr treatment (Fig. 4C). The expression levels of total p70S6K1 in adipocytes were not altered in response to SFN and/or Myr treatment (Fig. 4C).