Genistein

Fetal bovine serum (FBS), 100× penicillin, streptomycin, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Lonza (Verviers, Belgium). Genistein was obtained from Selleck (Shanghai, China). Genistein was dissolved in DMSO with a stock solution of 100 mM and diluted in culture medium to final concentrations (12.5–100 µM). Phosphatase and protease inhibitor cocktail set I and WST-8 buffer were purchased from Biotool (Shanghai, China). Enhanced chemiluminescence (ECL) detection buffer was purchased from Beyotime Biotechnology (Shanghai, China). Antibodies used in this study were purchased from Cell Signaling Technology (Danvers, MA, USA), and are listed in Table 1.

The 3T3-L1 cells were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS) in an incubator under an atmosphere of 5% CO₂ at 37 °C and cultured for 2 days until the cells grew to about 80%. The cells were induced to differentiate by incubation in a differentiation induction medium consisting of DMEM, 10% FBS, 10 mg/L insulin, 1 µM dexamethasone (DEX), and 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) for 2 days. The culture medium was then replaced with differentiation maintenance medium, consisting of DMEM, 10 mg/L insulin, and 10% FBS for 5 days, and the medium was changed once on day 2. Then the cells were harvested for the experiment.
To evaluate the effect of soy isoflavones on lipid accumulation and its suppression function, 25 μM of daidzein (Sigma-Aldrich, St. Louis, MO, USA) and 25 μM of genistein (Sigma-Aldrich) were added to the induction medium and differentiation medium. In the AMPK inhibition assay, 5 μM of Compound C (Selleck Chemicals, Houston, TX, USA) were added simultaneously with daidzein or genistein.

Hexaminolevulinate (HAL) hydrochloride, Ethylenediaminetetraacetic acid (EDTA) and phosphate-buffered saline (PBS) tablets were purchased from Sigma-Aldrich, Castle Hill,, NSW, Australia. Genistein, selumetinib, trametinib, U0126-EtOH, salicylic acid and deferiprone were obtained from Selleck, Scoresby, VIC, Australia.

Bortezomib and endocytosis inhibitors, including heparin, chlorpromazine, amiloride, dynasore, wortmannin, omeprazole, and genistein were purchased from Selleck Chemicals (Houston, TX, USA). 5-(N-Ethyl-Nisopropyl) amiloride (EIPA) was obtained from Cayman Chemical (Ann Arbor, MI, USA). Antibodies against flotillin-1 (Flot1, D2V7J, 18634T, 1:1000), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, D16H11, 5174S, 1:1000), calreticulin (D3E6, 12238T, 1:1000), caveolin-1 (CAV-1, D46G3, 3267T, 1:1000), and clathrin heavy chain (CLTC, D3C6, 4796S, 1:1000) were purchased from (Cell Signaling Technology, Danvers, MA, USA). Antibodies against dynamin-2 (DNM2, EPR9053, ab151555, 1:1000) and CD9 (EPR2949, ab92726, 1:2000) were bought from Abcam (Cambridge, United Kingdom). Anti-human CD63 antibody (Ts63, 10628D, 1:250) was obtained from Thermo Fisher Scientific (Waltham, MA, USA). IRDye 680RD or 800CW goat anti-mouse/rabbit IgG secondary antibodies (1:10000) were purchased from LI-COR Biosciences (Lincoln, NE, USA).

All reagents were of analytical grade and purchased from commercial sources. Correctors MCG1516A (#STK370345, Vitas-M laboratory, IL, USA) and VX-661 (#S7058, Selleckchem, TX, USA) were diluted in dimethyl sulfoxide (DMSO) and added to cells diluted in 1% fetal bovine serum (FBS, #10270106, Gibco) supplemented antibiotic-free medium. Other reagents (all from Sigma-Aldrich, MO, USA, unless otherwise stated) were also in DMSO solutions: forskolin (Fsk, #66575-29-9), genistein (Gen, #446-72-0), VX-770 (#1144, Selleckchem, Houston, TX, USA). The final concentration of each compound has been indicated in figure legends.