35 mm cell culture dishes

Cells were grown on 35-mm cell culture dishes with glass bottoms (NEST, Wuxi, China). Cells were fixed with 4% paraformaldehyde for 30 min. Fixed cells were washed and permeabilized with 0.1% Triton X-100 for 10 min. Cells were blocked with goat serum for 30 min and incubated with the anti-β-catenin antibody (1:100, 8480S, CST) overnight at 4 °C. After washing with PBS, the cells were subsequently incubated with Alexa Fluor^® 488-conjugated anti-rabbit IgG antibody (1:1000, ab150077, Abcam) for 1 h. The nuclei were stained with DAPI (62248, Thermo Scientific). Images were acquired using a Zeiss LSM710 confocal microscope (Zeiss, Germany) at 400× magnification.

Preadipocytes were inoculated in the 35 mm cell culture dishes (NEST Biotechnology, Wuxi, China). The cells were washed two times with phosphate-buffered saline (PBS) and fixed in 10% paraformaldehyde for 30 min. Subsequently, Oil Red O (NJBI, Nanjing, China) was mixed with deionized water at a rate of 3:2 and then added to the stained cell for 25 min. Finally, the cells were washed with PBS. Images were collected under an inverted microscope. Then, 2 mL of isopropyl alcohol was added into the cell culture dishes, and the cells were fully lysed and then transferred into the 96-well plate. The absorbance at 510 nm was measured by Thermo Scientific^TM Varioskan LUX. The absorbance was drawn and analyzed using GraphPad Prism 9 (GraphPad Software Inc., La Jolla, CA, USA). Moreover, the count of lipid droplets was measured using image-pro plus 6.0 software (Media Cybernetics).