Oil red o staining solution

AML12 cells were inoculated into 6-well culture plates and cultured for 24 h. The test wells were treated with the additional of 10 μL of compound 9, with a final concentration of 5 μM, 10 μM, and 20 Μm, and fenofibrate, with a final concentration of 20 Μm, respectively. Each sample had 3 duplicate wells, and 10 μL of blank matrix was added to the negative control wells and the OA-treated control wells for incubation for 2 h. Then, it was stimulated in the presence or absence of OA (500 μM) for 24 h to stimulate intracellular lipid accumulation. Oil red O staining solution (C0158S, Beyotime, Shanghai, China) was added to the treated cell samples for 10–20 min after washing, and hematoxylin staining solution was used for the nuclear staining. The results of the lipid accumulation were observed under a microscope.

3T3-L1 preadipocyte cell was got from Procell (Wuhan, China). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were bought from Solarbio (Beijing, China). Oil red O staining solution was purchased from Beyotime Biotechnology (Shanghai, China). 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (Dex), and insulin were obtained from Sigma-Aldrich (St. Louis, MO, United States). Sodium orthovanadate (Van) was purchased from Aladdin (Shanghai, China). Primer IRS, AKT, PI-3K, and GLUT4 were designed and synthesized by Thermo Fisher Scientific (Shanghai, China). Evo M-MLV RT Kit with gDNA Clean and TB Green TM Ex TaqTM II (Tli RNadeH Plus), Bulk kit were obtained from TaKaRa. Anti-IRS antibody, anti-phospho-IRS antibody, anti-AKT antibody, and anti-phospho-AKT antibody were bought from Cell Signaling Technology (Danvers, MA, United States). Anti-PI-3k antibody and anti-GLUT4 antibody were purchased from Abcam (Cambridge, United Kingdom).