Isopropanol

To investigate the inhibitory effects of GA on lipid accumulation during adipogenesis, 3T3-L1 cells were differentiated in 12-well plates with and without treatment with GA (300 and 600 μM) during every replacement of medium. After completing differentiation, the cells were rinsed with phosphate-buffered saline (PBS; Biosesang, Korea) and fixed with 0.4% formaldehyde solution (Junsei Chemical Co., Ltd., Japan; diluted in PBS) for 5 min. Cells were then washed with 60% isopropanol (Sigma–Aldrich Inc.) and fixed with 0.4% formaldehyde for 1 h. Formaldehyde was removed, and the cells were stained with 3.5 g/l of Oil Red O (Sigma–Aldrich Inc.; diluted in isopropanol) for 10 min. The stained cells were rinsed thrice with distilled water and observed using a phase-contrast microscope. To quantify the stained Oil Red O dye in lipid droplets, the dye was eluted using 100%isopropanol for 5 min. The absorbance of the eluted dye was measured at 540 nm using a VersaMax microplate reader (Molecular Devices).

For adipogenic differentiation, hASCs in adipogenic and control media were fixed with 4.0% paraformaldehyde at day 7 to assess intracellular lipid vesicles using Oil red O staining [31 (link)]. Briefly, 60% isopropanol (Sigma-Aldrich) was added to the wells and incubated for 5 min at RT. After removing the isopropanol, cells were stained with 0.3% Oil red O (Sigma-Aldrich) for 15 min. After washing, hematoxylin was added for 1.0 min to counterstain the cells before imaging. The stain was then extracted using 99% isopropanol and quantified using the microplate reader (Molecular Devices, San Jose, CA, USA) at 530 nm absorbance.

Microfluidic devices were fabricated using cell cast PMMA (poly(methyl methacrylate)) sheets (Weatherall Equipment, Wendover, UK) with a culture chamber of 2.5  $\mathrm{\mu }$ L volume. The devices were designed using AutoCAD 2016 and consist of 3 PMMA sheets. The sheet thickness was 200 $\mathrm{\mu }$ m for both the top and the bottom layers and 500 $\mathrm{\mu }$ m for the middle layer. PMMA sheets were cut to desired size and functionality (holes or channels) using a laser engraving system (L3040 from HPC Laser LTD, Elland, UK). The sheets were bonded using the protocol described in^{39 (link)}. Briefly, the cut sheets were washed with isopropanol (Sigma-Aldrich); ethanol (Sigma-Aldrich); and isopropanol again. The sheets were air-dried and the bonding surfaces were exposed to oxygen plasma for 80 secs using a Bd-20AC laboratory corona treater (Electro-Technic Products, Chicago, IL, USA). 2.5% of dibutyl phthalate (DBP) (Sigma-Aldrich) in isopropanol was used as a plasticiser. A single coat of DBP solution of 18 $\mathrm{\mu }$ L was applied to bonding surfaces. PMMA sheets were kept in an oven at 65 ${}^{\circ }$ C for 15 minutes before bonding them under 20 MPa of pressure at 85 ${}^{\circ }$ C for 15 minutes using a hydraulic press (Specac Ltd, Kent, United Kingdom).

The complete thoracic and abdominal aorta was harvested from mice and stored in PBS at 4 °C. Prior to staining, excess adipose fat was removed. Aortas were rinsed in ddH₂0 before soaking in 60% isopropanol (Sigma) for 10 min. Tissues were stained with Oil Red O (Sigma) for 30 min and further washed in 60% isopropanol for 2 min. Aortas were imaged using a Nikon Eclipse TE2000-U microscope. Oil Red O content was quantitated using ImageJ software (NIH, Bethesda, USA) and expressed as a percentage of the total tissue area.

Freeze-dried liver samples were used for the determination of total cholesterol, HDL- cholesterol, LDL-cholesterol and TAG. Lipids were extracted using a modified method with low-toxicity solvent⁽²⁸ (link)⁾. Briefly, 20 mg were dissolved in a 3:2 solution containing hexane (Sigma-Aldrich), isopropanol (Merck), with 0·005 % (v/w) 2,6-di-tert-butyl-4-metylphenol (Merck) before being centrifuged, and the supernatants were evaporated under N₂ flow at room temperature. The dried lipid extracts were re-dissolved in 1 ml of isopropanol containing 1 % (v/v) Triton X100 (Sigma-Aldrich). Total cholesterol and TAG in liver and serum were determined spectrophotometrically using Infinity Cholesterol/Triglyceride Liquid Stable Reagent (Thermo Scientific), while liver HDL and LDL were measured using the reagents HDL-Cholesterol Plus and LDL-Cholesterol (Thermo Scientific).