Fig 2: Tributyltin Chloride Induces Varied Adipogenic Activities Based on Induction Time.ATCC 3T3-L1, Zenbio 3T3-L1, and OP9 cells were differentiated as described in Methods and assessed for adipocyte differentiation (Nile Red staining of lipid accumulation) and cell proliferation (Hoechst staining) at various times after initiation of differentiation. Percent raw triglyceride accumulation per well relative to maximal rosiglitazone response for tributyltin chloride (TBT) at 7 days (A), 10 days (B), and 14 days (C). Increase (cell proliferation) or decrease (potential cytotoxicity) in DNA content relative to vehicle control for TBT at 7 days (D), 10 days (E), and 14 days (F). Percent normalized triglyceride accumulation per cell (normalized to DNA content) for TBT at 7 days (G), 10 days (H), and 14 days (I). Triglyceride accumulation responses are provided as relative activity to maximal rosiglitazone. Data presented as mean ± SE from three independent experiments. *Indicates lowest concentration with significant increase in triglyceride over vehicle control, p < 0.05, as per linear mixed model in SAS 9.4.

Fig 3: GW9662 Induces Varied Adipogenic Inhibition Based on Induction Time.ATCC 3T3-L1, Zenbio 3T3-L1, and OP9 cells were differentiated as described in Methods and assessed for adipocyte differentiation (Nile Red staining of lipid accumulation) and cell proliferation (Hoechst staining) at various times after initiation of differentiation. Percent raw triglyceride inhibition of half maximal rosiglitazone per well for GW9662 at 7 days (A), 10 days (B), and 14 days (C). Increase (cell proliferation) or decrease (potential cytotoxicity) in DNA content relative to vehicle control for GW9662 at 7 days (D), 10 days (E), and 14 days (F). Percent normalized triglyceride accumulation per cell (normalized to DNA content) for GW9662 at 7 days (G), 10 days (H), and 14 days (I). Data presented as mean ± SE from three independent experiments. *Indicates lowest concentration with significant increase in triglyceride over vehicle control, p < 0.05, as per linear mixed model in SAS 9.4.

Fig 4: Mechanistic Receptor Controls Induce Varied Adipogenic Activities Between Cell Lines.ATCC 3T3-L1, Zenbio 3T3-L1, and OP9 cells were differentiated as described in Methods and assessed for adipocyte differentiation (Nile Red staining of lipid accumulation) and cell proliferation (Hoechst staining) following seven days (OP9) or ten days (3T3-L1) of treatment with mechanistic receptor control ligands. Percent raw triglyceride accumulation per well relative to maximal rosiglitazone response for rosiglitazone (RSG, PPAR? agonist), GW3965 (liver X receptor, LXR, agonist), dexamethasone (glucocorticoid receptor, GR, agonist), 1–850 (thyroid receptor, TR, antagonist), flutamide (androgen receptor, AR, antagonist), and LG100268 (retinoid X receptor, RXR, agonist) in ATCC 3T3-L1 cells (A), Zenbio 3T3-L1 cells (B), and OP9 cells (C). Increase (cell proliferation) or decrease (potential cytotoxicity) in DNA content relative to vehicle control for test chemicals in ATCC 3T3-L1 cells (D), Zenbio 3T3-L1 cells (E), and OP9 cells (F). Percent normalized triglyceride accumulation per cell (normalized to DNA content) for test chemicals in ATCC 3T3-L1 cells (G), Zenbio 3T3-L1 cells (H), and OP9 cells (I). Responses are provided as relative activity to maximal rosiglitazone. Data presented as mean ± SE from three independent experiments. *Indicates lowest concentration with significant increase in triglyceride over vehicle control, p < 0.05, as per linear mixed model in SAS 9.4.

Fig 5: Ephx1 deficiency suppresses adipocyte differentiation of 3T3-L1 cells and alters insulin signaling.Data were obtained in 3T3-L1 pre-adipocytes from ATCC, 3T3-L1 cells with a CRISPR-Cas9-mediated Ephx1-knockout (KO), and 3T3-L1 cells transfected with a Cas9/scramble gRNA plasmid corresponding to control (CTL) cells. (A) Timeline representation of the 3T3-L1 pre-adipocyte differentiation process using a hormonal cocktail. Dexa: dexamethasone; IBMX: 3-isobutyl-1-methylxanthine; D0–D12: Day 0 to Day 12. (B) Validation of Ephx1 KO in 3T3-L1 pre-adipocytes and study of its expression during adipocyte differentiation. Numbers on the left correspond to molecular weight markers (kDa). Western blot images are representative of three independent experiments. (C) Adipocyte differentiation assessed by Oil Red O lipid staining. 3T3-L1 pre-adipocytes were studied during adipocyte differentiation for 12 days. First and third lines: Pictures of dishes stained by Oil Red O. Images are representative of three independent experiments. Second and fourth lines: representative images of fluorescence microscopy after staining of intracellular lipids (Oil Red O, red) and nuclei (DAPI, blue). Images are representative of five independent experiments. (D) Quantification of Oil Red O fluorescence normalized to DNA content (DAPI). Results are expressed as means ± SEM of five independent experiments. ****p<0.0001. p-values were determined by analysis of variance (ANOVA) with Kruskal–Wallis post hoc multiple comparison test. (E) Protein expression of adipocyte markers obtained by western blotting during in vitro adipocyte differentiation of 3T3-L1 pre-adipocytes. Numbers on the left correspond to molecular weight markers (kDa). Western blot images are representative of three independent experiments. PPARγ: peroxisome proliferator-activated receptor gamma; C/EBPα: CCAAT/enhancer-binding protein alpha; SREBP-1c: sterol regulatory element-binding protein-1c; FAS: fatty acid synthase. (F) Activation of insulin signaling in 3T3-L1 pre-adipocytes after 10 days of adipocyte differentiation. The 3T3-L1 cells from ATCC, CTL, and Ephx1-KO cells were deprived of serum for 6 hr, stimulated with 20 nM insulin for 5 min or left untreated, and subjected to immunoblotting with antibodies against total and phospho-insulin receptor β-subunit (IRβ), insulin receptor substrate-1 (IRS1), AKT, and extracellular-regulated kinase (ERK)1/2. Numbers on the left correspond to molecular weight markers (kDa). Western blot images are representative of three independent experiments.