Fig 2: Temporal expression of transcription factors and terminal differentiation markers after exposure to Firemaster® 550 and its components.mRNA expression levels were determined in 3T3-L1 preadipocytes 2, 4, 6, and 9 days post-treatment with the differentiation cocktail consisting of IBMX, insulin, and either 100 µM FM550, 10 µM IPTP, 20 µM TPP, or 20 µM TBPH. After the indicated time-points, RNA was extracted and reverse transcribed, and we measured the levels of (A) Ppar?, (B) Cebpa, (C) aP2, (D) Lpl, and (E) Plin, which were normalized to ß-actin levels and relative to time-matched vehicle control (MI). Data represent the mean ± S.E.M (n = 3–5). * P<0.05 relative to time-matched vehicle control using a one-way ANOVA followed by Tukey’s post-hoc analysis.

Fig 3: The components IPTP and TPP induce endogenous expression and recruitment of PPARγ to the aP2 enhancer region.3T3-L1 preadipocytes cells exposed to the differentiation cocktail for 6 days were used for the ChIP assay to measure the endogenous expression and recruitment of PPARγ to the aP2 enhancer region by RT-PCR. Positive controls dexamethasone (DEX) and troglitazone (TROG) were used and depicted in (A). (B) PPARγ recruitment to the aP2 enhancer region after exposure to 20 μM TPP and 10 μM IPTP was determined. Data represents the mean of n = 6 independent replicates ± SEM. Data were relative to 100% input DNA and significance was determined relative to control PPARγ recruitment using a one-way ANOVA followed by Tukey’s post-hoc analysis (*P<0.05).

Fig 4: The effects of selective PPAR? antagonist GW9662 on aP2 mRNA expression and lipid accumulation in the presence of troglitazone, TPP, IPTP, and TBPH.Murine 3T3-L1 preadipocytes were induced to differentiate in the presence of 500 µM IBMX (M), 100 nM insulin (I), supplemented with indicated treatments: 5 µM troglitazone (TROG), 0.25 µM dexamethasone (DEX), 20 µM TPP, 10 µM IPTP, or 20 µM TBPH with either solvent control (Veh) or PPAR? inhibitor GW9662 (GW) twice daily. (A) At day 6 of differentiation mRNA expression levels of aP2 were quantified by RT-qPCR normalized to MI control conditions. (B-E) At days 2, 4, 6, and 9 lipid accumulation was visualized using Nile Red staining and then quantified. Lipid accumulation was normalized to DAPI staining. Data represent mean ± SEM for n = 3 independent experiments. * denotes p<0.05 when comparing treatment to vehicle (MI) conditions (A). # denotes p<0.05, ## denotes p<0.01, and ### denotes p<0.001 when comparing to GW9662 at corresponding day of differentiation (B-E). Statistical analysis was performed by one-way ANOVA with Tukey’s post-hoc tests.

Fig 5: The ability of FM550 and its components to activate the PPRE-dependent aP2 reporter construct which requires direct PPAR? activation.COS-7 cells were transfected as described in the Materials and Methods with pcDNA-mPPAR?, pcDNA mRXR, aP2 enhancer-luciferase, and pCMV-RL and treated with increasing amounts of the positive control (A) Troglitazone (0.02–5 µM), (B) FM550 (0.1–200 µM), or the components (C) IPTP (0.1–20 µM) (D) TPP (0.1–20 µM) (E) TBB (0.1–20 µM) (F) TBPH (0.1–20 µM) (G) DPP (0.1–20 µM). Twenty-four hours after treatment reporter gene activity was determined. Data represent the mean ± S.E.M of three independent experiments. Significantly different (*P<0.05) reporter gene activity was analyzed relative to transfected vehicle-treated cells using a one-way ANOVA followed by Tukey’s post-hoc analysis.