**2. Materials and Methods**

### *2.1. Plant Material and Isolation of CCT*

Saffron was obtained from the "Agrícola Técnica de Manipulación y Comercialización" company (Minaya, Albacete, Spain) during the 2014–2015 harvest. These dried stigmas belonged to the Protected Designation of Origin (PDO) "Azafrán de La Mancha", which complies with ISO 3632:2011 (Category I) and guarantees their origin and freedom from fraud. Saffron with a very low moisture level was stored in the dark at 4 ◦C until further use.

CCT was obtained via the hydrolysis of aqueous solutions of saffron acquired using a protected internal method of the "Verdú Cantó Saffron Spain" company (Novelda, Alicante, Spain) [22]. CCT purity was checked through the reverse-phase (RP)-HPLC–diode array detection (DAD) technique according to [32]. Twenty microliters of aqueous extracts of CCT were filtered through a syringe with a polytetrafluoroethylene (PTFE) filter, 0.45 μm pore size (Millipore, Bedford, MA, USA), and injected into an Agilent 1200 chromatograph (Palo Alto, CA, USA). Chromatographic determination was achieved using a Phenomenex Luna C18 column (150 × 4.6 mm, 5 μm) (Le Pecq CEDEX, France) equilibrated at 30 ◦C. Acetonitrile (ACN) and Milli Q water (mQW) were used as the mobile phase at a flow rate of 0.8 mL/min. HPLC-grade ACN was obtained from Panreac® (Barcelona, Spain) and ultrahigh-purity water mQW was produced using a Milli-Q system (Millipore, Danvers, MA, USA). The elution gradient was set up for the ACN solvent as follows: 20%, 0–5 min; 20–80%, 5–15 min; 80%, 15–18 min; and 20%, 18–30 min. The DAD detector (Hewlett Packard, Waldbronn, Germany) was set to 440 nm for *cis*/*trans*-CCT detection. The chromatographic purity of *cis*/*trans*-CCT according to HPLC–DAD at 440 nm was 99% (86% *trans*-CCT, retention time: 16.64 min; 13% *cis*-CCT, retention time: 17.86 min). CCT was stored at −20 ◦C until further use. Before using CCT to carry out the experiments, purity was determined again obtaining the same chromatographic purity before it was employed.

### *2.2. 3T3-L1 Cell Culture and Adipocyte Di*ff*erentiation*

The cell line of embryonic fibroblasts, 3T3-L1, was acquired in 2016 from the ATCC (American Type Culture Collection, Manassas, VA, USA). 3T3-L1 preadipocytes were cultured, maintained, and differentiated according to the supplier's instructions. In all experiments, cells were used within the sixth passage. Briefly, 3T3-L1 cells were expanded in a 75 cm<sup>2</sup> flask at 37 ◦C under a humidified 5% CO2 atmosphere in preadipocyte expansion medium (EM; Dulbecco's modified Eagle's medium (DMEM, 90%) supplemented with l-glutamine (1%), penicillin/streptomycin (0.5%), and inactivated bovine calf serum (BCS, 10%). When the cells reached 70–80% confluence, they were seeded on sixor 96-well sterile plates and grown in EM for 48 h or until the culture reached 90% confluence. Then, differentiation of adipocytes was induced in the absence (as a control of differentiation) or presence of CCT (1 or 5 μM). Stimulating and inhibiting controls of differentiation were also established with rosiglitazone (10 μM, an agonist of PPARγ that activates adipogenesis [33,34]) or genistein (12.5 μM, an isoflavone that inhibits adipogenesis [35,36]), respectively. Genistein at this concentration inhibits lipid accumulation while preserving the viability of preadipocytes [36]. For this, post-confluent cells were treated for 48 h with differentiation medium (DM). The DM was prepared using the same components as the EM, instead replacing CBS with inactivated fetal bovine serum (FBS, 10%) and adding an adipogenic cocktail (AC), containing substances to induce differentiation (0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 0.25 μM dexamethasone, and 1 μg/mL insulin (INS)). The DM was subsequently replaced with adipocyte maintenance medium (MM), which was composed of DMEM containing 1 μg/mL INS and 10% FBS. Cells were maintained in MM for 6 days, with the medium replenished every 2 days. At this point, the cells developed large lipid droplets and were considered mature adipocytes (Figure 1).

**Figure 1.** Schematic representation of the differentiation procedure to analyze the effects of crocetin (CCT). Fibroblasts achieved confluence after 48 h of treatment with expansion medium (EM). Then, the differentiation of confluent fibroblasts was induced with differentiation medium (DM; with or without CCT (1 or 5 μM)) for 48 h. Subsequently, preadipocytes were maintained with maintenance medium (MM) for 6 days. ID, initial differentiation; FD, final differentiation.

To check the effect of CCT on the early and late genes of differentiation, cells were collected at two time-points: 48 h after induction (initial differentiation, ID) and 6 days after differentiation (final differentiation, FD).

Two concentrations of CCT (1 and 5 μM) were tested in this work. This choice was based on our experience in previous studies [22] and on the work of Chryssanthi et al. [37]. These were prepared from a stock CCT solution, dissolved in sterile dimethyl sulfoxide (DMSO) and added to DM (the final concentration of DMSO in the culture medium was 0.001% (*v*/*v*)). Rosiglitazone and genistein stock solutions were also prepared in DMSO and added to the DM to reach a working concentration (the final volume of DMSO in the well was 0.001% (*v*/*v*)). The differentiation of adipocytes was also carried out in the presence of sterile DMSO (0.001%) as the solvent control. DMSO is usually well tolerated with no observable toxic effects on cells at a 0.1% final concentration. This compound is widely used as a solvent for various pharmacological agents at concentrations of 0.05–1.5% [38].

### *2.3. Quantification of the Intracellular Fat by Oil Red O Staining*

Cells di fferentiated in 96-well sterile plates (cellular density, 4 × 10<sup>3</sup> cell/well) were stained with Oil Red O (OR) at the FD time-point, according to the method developed by Kraus and colleagues [39] with slight modification. Oil Red O is a dye that strongly stains lipids, specifically triacylglycerol, often used for the quantitative analysis of adipocyte di fferentiation [39].

The OR stock solution was prepared the day before as follows: 0.2 g OR was dissolved in 100 mL of isopropanol for 24 h at room temperature under agitation. The OR working solution was prepared by mixing six parts of OR stock solution and four parts of double-distilled water (ddH2O). The solution was filtered through a two-layer Whatman paper to remove any precipitate.

To stain cells with the OR working solution, they were first washed three times with phosphatebu ffered saline (PBS) and fixed in 4% formaldehyde for 1 h at room temperature while avoiding any shaking of the plate. Formaldehyde was removed, and the cells were washed once with cold PBS and air-dried for 10 min. The freshly prepared OR working solution was added to the plates to cover the cell surface. After 10 min, the solution was aspirated, and the cells were washed three times with cold PBS and air-dried for 15 min. OR was eluted with 100% isopropanol for 10 min, and absorbance was measured using a spectrophotometer (ASYS UVM 340, Cambridge, United Kingdom, Microplate Readers) at 450 nm. Data obtained from at least 10 replicates of each condition from three independent experiments were used for analysis. The amount of color produced is directly proportional to the amount of intracellular fat.

Reagents, unless specified otherwise, were acquired from Sigma-Aldrich.

### *2.4. Determination of the Number and Size of Lipid Droplets*

The number and size distribution of lipid droplets were evaluated by Wimasis (Edificio Centauro, 14014 Córdoba, Spain) using a WimLipid image analysis software. For this, photomicrographs (20X) of the wells at the FD time-point were taken using a phase-contrast microscope (Olympus 1X51). Parameters such as the circularity, convexity, and elongation were included in the analysis to discriminate drops. The following criteria were used: area ≥ 10 pixels (Px); circularity > elongation; convexity > 0.95. Drops that failed to meet these criteria were removed.

### *2.5. Quantification of Cellular Viability*

### 2.5.1. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Assay

To perform the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cells were grown on 96-well sterile plates at a cellular density of 4 × 10<sup>3</sup> cell/well. The MTT viability assay was carried out as previously described [40] with slight modification. The MTT assay measures mitochondrial activity in metabolizing cells and, therefore, can be used as an approximate measurement of cell viability. The assay relies on the reduction of MTT, a yellow water-soluble tetrazolium dye, primarily by mitochondrial dehydrogenases, to purple-colored formazan crystals. An MTT stock solution in PBS was freshly prepared and assessed in all experimental conditions. At the FD time-point, cells were washed with red phenol-free DMEM without FBS. Then, 100 μL of MTT solution (red phenol-free DMEM with MTT 0.5 μg/μL) was added to each well, mixed gently, and incubated for 45 min at 37 ◦C. Media were immediately aspirated and discarded; then, in order to solubilize formazan crystals, 100 μL of DMSO was added to each well before gently stirring for 3–5 min. Absorbance was determined spectrophotometrically at 570 nm using a reference wavelength of 630 nm (ASYS UVM 340, Cambridge, United Kingdom, Microplate Readers). The color intensity is directly proportional to the number of viable cells. Data obtained from at least 10 replicates in each experimental condition from three independent experiments were used for analysis. Absorbance was measured for wells containing the control di fferentiated cells (DM-di fferentiated cells) and the DM + CCT (1 or 5 μM)-di fferentiated cells. Furthermore, di fferentiated cells in the presence of solvent (DM + DMSO (0.001%)) and an activator (DM + rosiglitazone 10 μM) or inhibitor (DM + genistein 12.5 μM) of adipogenesis were also measured.
