Isopropanol

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.

ST2 cells were plated in 6-well plates at a density of 1 × 10⁴ cells/cm² in normal culture media. After 24 h, the media were replaced with media supplemented with 0.5 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma), 1 nM dexamethasone (Sigma), 10 µg/mL insulin (Sigma), and 10% (v/v) serum from control or LLC-bearing mice. After incubation for three days, the cells were washed with PBS twice and fixed with 4% PFA in PBS for 1 h. The fixed cells were washed with water twice and incubated with 60% isopropanol (Sigma) for 5 min. Then, the cells were stained with 0.3% Oil Red O (Sigma) for 30 min. After washing the plate twice with water, images of the stained cells were captured using an Eclipse TS100 light microscope (Nikon). To quantify the Oil Red O stained lipid drops, the stained cells were rinsed with 60% isopropanol and subsequently dried. The stains were eluted with 1 mL of 100% isopropanol for 10 min at 25 °C and the optical density was measured at 540 nm using an absorbance plate reader (Spectramax190; Molecular Devices; Thermo Fisher Scientific Inc., Waltham, MA, USA). If required, 100 nM ruxolitinib or 10 µM mifepristone was added daily for three days during which the ST2 cells underwent adipogenic differentiation on incubation with the mice serum.