ID Bioinformatics Sources six.eight database (http://david.ncifcrf.gov/home.jsp (accessed on 19 August 2021)) [49]. The false discovery rate (FDR) error manage method (FDR 0.05) was utilized to correct the p-value. Lastly, a threshold value of p 0.05 was set and signaling pathways have been obtained. The KEGG pathway enrichment analysis results had been visualized employing ImageGP (EHBIO Gene Technology, Beijing, China) (http://ehbio/ImageGP (accessed on 19 August 2021)). 2.1.5. Building and Analysis of Compound arget athway (C ) Networks The targets related with this pathway were obtained from the KEGG pathway enrichment evaluation. Cytoscape (three.eight.two) (NRNB, Bethesda, MD, USA) was made use of to visualize and analyze the C network. two.two. Cell Culture and Adipogenic Differentiation The mouse preadipocyte cell line (3T3-L1) was obtained in the American Kind Culture Collection (Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s medium (DMEM; Cellgro, Manassas, VA, USA) containing ten bovine calf serum (BCS; Gaithersburg, MD, USA) and 1 penicillin/streptomycin antibiotics (P/S; Gaithersburg, MD, USA). To induce adipogenesis, 3T3-L1 CXCR4 Agonist medchemexpress preadipocytes (four 104 cells/well)Biomolecules 2021, 11,four ofwere grown within a 24-well plate for two days, then the culture medium was replaced using the adipogenic differentiation medium containing 0.four /mL dexamethasone (DYRK2 Inhibitor Synonyms SigmaAldrich, St. Louis, MO, USA), 10 fetal bovine serum (FBS; Gaithersburg, MD, USA), 1-methyl-3-isobutylxanthine (IBMX; Sigma-Aldrich, St. Louis, MO, USA), 1 P/S antibiotics, and 5 /mL insulin (Sigma-Aldrich, St. Louis, MO, USA). Immediately after incubation for two days, the culture medium was replaced with DMEM supplemented with 10 FBS, five /mL insulin, and 1 P/S antibiotics just about every 2 days. Finally, the culture medium was replaced with DMEM containing ten FBS and 1 P/S antibiotics, which was changed just about every two days, as previously described [50]. Hispidulin (5, ten, 20, and 40 ) and p-synephrine (5, 10, 20, and 40 ) have been added individually or in mixture inside the culture medium for the duration of adipogenic differentiation. Hispidulin (98 ) and p-synephrine (98 ) have been purchased from Sigma-Aldrich (St. Louis, MO, USA). two.three. Measurement of Cell Viability The viability of 3T3-L1 preadipocytes was assessed working with a tetrazolium salt (WST-1)-based colorimetric assay kit (Ez-Cytox Cell Viability Assay Kit; Daeil Lab Service, Seoul, Korea). The 3T3-L1 preadipocytes (4 104 cells/well, 96-well plate) were grown in ten BCS and 1 P/S antibiotics for 24 h, then treated with hispidulin (5, 10, 20, and 40 ) and p-synephrine (5, 10, 20, and 40 ) individually or in mixture. After therapy for 24 h, EZ-Cytox reagent was added, and also the 3T3-L1 preadipocytes had been additional incubated for 40 min. The spectrophotometric absorbance was measured working with a PowerWave XS microplate reader (BioTek Instruments, Winooski, VT, USA) at 490 nm, as previously described [51]. 2.4. Oil Red O Staining On day eight, differentiated cells had been fixed with 4 paraformaldehyde resolution (SigmaAldrich, St. Louis, MO, USA) for 1 h and stained with Oil Red O remedy containing 0.5 Oil Red O (ORO; Sigma-Aldrich, St. Louis, MO, USA), 40 distilled water (DW), and 60 isopropanol (Sigma-Aldrich, St. Louis, MO, USA) for 1 h. Following washing with DW, lipid droplets stained with ORO were imaged under an inverted microscope at 20magnification and eluted with 100 isopropanol. The spectrophotometric absorbance was measured on a PowerWave XS microplate reader at 540 nm, as pre