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) Pparγ, (B) Cebpα, (C) aP2, (D) Lpl, and (E) Plin, which were normalized to β-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: Protein expression in the mature adipocyte following exposure to Firemaster® 550 and its components.Representative immunoblots depicting the ability of (A) Firemaster® 550 (0.1–200 µM), and the components (C) IPTP (0.1–10 µM) and (E) TPP (0.1–20 µM) at inducing the expression of aP2, LPL, PLIN following 9 days of treatment. ß-actin was used as the loading control. Quantification of aP2, LPL, and PLIN protein expression levels after (B) FM550 (D) IPTP (F) TPP treatment (n = 3–5) using ImageLab software (BioRad) and ß-actin as loading control. (G) TBPH, TBB DPP treatments at day 9. The positive controls dexamethasone (MID) and troglitazone (MIT) are shown. Blots are representative of at least three independent experiments. *P<0.05 compared to MI control using a one-way ANOVA followed by Tukey’s post-hoc analysis. Data are expressed as mean ± SEM.

Fig 4: Obesity in Par-4-/- mice is rescued by deletion of C3. (A) Par-4 and C3 double knockout (DKO) mice are not obese. Weight of age-matched DKO (n=13, 10, 8 and 4 for each time point respectively) and Par-4+/+ (n=27, 16, 9, 5 for each time point respectively) male mice was determined over the course of 7 months; representative images of Par-4+/+ and DKO at 5-6 months are shown. (B) DKO mice do not show increased fat accumulation. Six-month-old DKO (n=8) and Par-4+/+ (n=7) male mice were examined for fat mass by Echo-MRI and adipocyte hypertrophy. (C) Triglyceride absorption is similar in DKO, C3KO and Par-4+/+ mice. DKO (n=5-6), C3-/- (n=4) and Par-4+/+ (n=11) male mice were fasted overnight and injected intraperitoneally with tyloxapol, 30 min before oral gavage of olive oil. Plasma was collected at the indicated time intervals after fat load, and triglycerides were quantified. Plasma was also subjected to western blot analysis for ApoB48. ApoB48 levels in the plasma were normalized to albumin in corresponding Coomassie blue gels, and fold change at each time point is shown (n=4). (D) LPL is upregulated in the adipose tissue of AKO and Par-4-/- mice. LPL protein levels were determined by western blot analysis in visceral adipose tissue and heart from AKO, Par-4-/- and Par-4+/+ mice (n=3). (E) LPL is not increased in the adipose tissue of DKO mice. LPL protein levels were determined by western blot analysis in visceral adipose tissue and heart from Par-4+/+ (n=4) and DKO (n= 4) male mice. LPL levels were normalized to β-tubulin levels and fold change was calculated. Mean ± SEM, *P < 0.05, **P < 0.01, by the Student’s t-test. Also see Figures S6 , S13 .

Fig 5: Exoc5-SMKO mice had increased hepatic lipid accumulation, yet no difference in insulin-sensitive fatty acid transporter CD36 protein levels was detected.A, representative images of control and Exoc5-SMKO liver samples stained with Oil Red O (left panels). Quantification of the ratio of Oil Red O-stained areas in liver tissues (right panel). B, images of Western blot analysis (left panel) and quantification (right panel) of CD36. CD36 levels in B as well as LPL and FASN levels in Fig. S5 were sequentially analyzed on a single Western blot, and their respective signal intensities were normalized using the same ßactin loading control measurements. Thus we show the same ßactin loading control images in Fig. S5,B and D as well. Black scale bars, 100 µm, Error bars = standard deviation, **p = 0.01.