The effect of exogenous palmitic acid on 3T3-L1 preadipocytes.

<p>(A) 3T3-L1 preadipocytes were treated with α-mangostin and palmitic acid at various concentrations (α-mangostin: 0, 6, 12, 18, 30 µM; palmitic acid: 0, 25, 50, 100 µM) for 24 h. Cell viability was determined by MTT colorimetric assay. Assays were performed on eight replicates for each treatment. Results are expressed as percentages of cell viability as compared with untreated control (means ± S.D., n = 8). The experiment was repeated in twice. (B) The 3T3-L1 preadipocytes were treated with α-mangostin and palmitic acid at various concentrations (α-mangostin: 0, 30 µM; palmitic acid: 0, 25, 50, 100 µM) for 24 h. And then the amount of intracellular fatty acid was determined by Fatty Acid Assay Kit. Data are expressed as means ± S.D. (<i>n</i> = 3). * <i>p</i><0.05 different from respective control; ** <i>p</i><0.01 significantly different from respective control.</p

Inhibitory effect of α-mangostin on intracellular lipid accumulation.

<p>The intracellular lipid content was measured by <i>Oil Red O staining</i> as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033376#s4" target="_blank">Materials and Methods</a>. (A) Cells were photographed at 40× magnification. The experiment was performed on three replicates for each treatment. Representative images are shown. (B) Quantitative analysis of lipid accumulation. Each value is expressed as means ± SD (<i>n</i> = 3). * <i>p</i><0.05 different from control (0 µM); ** <i>p</i><0.01 significantly different from control (0 µM).</p

Effect of α-mangostin on FAS activity in 3T3-L1 preadipocytes.

<p>3T3-L1 preadipocytes were treated with α-mangostin at the indicated concentrations for 24 h. FAS specific activity was determined by <i>Cell FAS activity assay</i>. Data are expressed as means ± S.D. (<i>n</i> = 3). * <i>p</i><0.05 different from control (0 µM); ** <i>p</i><0.01significantly different from control (0 µM).</p

Inhibitory effects of α-mangostin on FAS activities.

<p>(A) and (B) Lineweaver-Burk plots for inhibition of FAS overall reaction: (A) Acetyl-CoA was the variable substrate. The concentrations of α-mangostin were 0 µM (⧫); 5 µM (•); 10 µM (▴); 15 µM (<b>+</b>). (B) Malonyl-CoA was the variable substrate. The concentrations of α-mangostin were 0 µM (⧫); 5 µM (•); 10 µM (▴); 15 µM (<b>+</b>). (C) Time courses of the overall reaction in the presence of α-mangostin. The reaction was determined by slow-binding inhibition assay. (D) Inhibition of FAS overall reaction and some partial reactions of FAS.</p

Effect of α-mangostin on proliferation of 3T3-L1 preadipocytes.

<p>(A) Cells were incubated with 0–36 µM α-mangostin for 24 h at 37°C in humidified 5% CO₂ incubator. (B) Cells were incubated with 0, 6, 12, 24, 30 µM α-mangostin for 6–24 h at 37°C in humidified 5% CO₂ incubator. Assays were performed on eight replicates for each treatment. Results are expressed as percentages of cell viability as compared with untreated control (means ± S.D., n = 8). The experiments were repeated in twice.</p

α-mangostin-induced 3T3-L1 preadipocytes apopotosis.

<p>3T3-L1 preadipocytes were treated with α-mangostin at the indicated concentrations for 24 h. (A) Effect of α-mangostin on cell membrane permeability: original magnification, ×40; exposure times: 20s; (B) Effect of α-mangostin on nuclear chromatin morphology with Hoechst 33258 staining: original magnification, ×200; exposure times: 100s. (C) Effect of α-mangostin on mitochondria membrane potential (ΔΨm) with JC-1 staining: original magnification, ×40; exposure times: 100 s. B. field, bright field; H.33258, Hoechst 33258; JC-1, 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolcarbocyanine iodide. The experiments were performed on four replicates for each treatment. Representative images are shown.</p

Effect of α-mangostin on cell viability of 3T3-L1 cells in different stages of adipocyte differentiation.

<p>3T3-L1 cells were exposed to various concentrations of α-mangostin at various time points (d₂, d₄ or d₈) for 24 h, then the cell viability were measured by MTT assay. Assays were performed on four replicates for each treatment. Results are expressed as percentages of cell viability as compared with each untreated control (means ± S.D., <i>n</i> = 4). The experiment was repeated in triplicate. Early stage: d₀-d₂; Middle stage: d₂-d₄; Late stage: d₄-d₈.</p

Effect of α-mangostin on lipolysis and FAS activity in 3T3-L1 mature adipocytes.

<p>3T3-L1 mature adipocytes were treated for at the indicated concentrations for 24 h, and then the intracellular lipid content, cell FAS activity were measured and cell sizes were compared. (A) Cells were photographed at 40× magnification. The experiment was performed on three replicates for each treatment. Representative images are shown. (B) Cells were photographed at 200× magnification. The experiment was performed on three replicates for each treatment. (C) Reduction of FAS activity. Data are expressed as means ± S.D. (<i>n</i> = 3).(D) Quantitative analysis of lipid lipolysis. Each value is expressed as means ± SD (<i>n</i> = 3). * <i>p</i><0.05 different from control (0 µM); ** <i>p</i><0.01 significantly different from control (0 µM).</p