Rar β

Immunocytochemical staining was conducted on cell-bearing coverslips by the method described elsewhere [15 (link)]. The antibodies used were: Cyclin D1 (Bioss Inc., Beijing, China; 1:100), Tg (Bioss. Inc., Beijing, China; 1:200), E-cadherin (Bioss. Inc., Beijing, China; 1:100), RAR-β (Bioss. Inc., Beijing, China; 1:150), and PPAR-β/δ (Bioss. Inc., Beijing, China; 1:200). Cells incubated in 0.2% DMSO-containing medium were used as a control. The color reaction was performed by using 3,3′-diaminobenzidine tetrahydrochloride (DAB) after the binding of the primary antibody (Vector Laboratories, Burlingame, CA, USA). For double immunofluorescent staining (IF), mouse anti-CRABP2 and rabbit anti-FABP5 were employed in the working concentrations of 1:120. Briefly, the cell-bearing coverslips were washed with phosphate-buffered solution (PBS, pH 7.4), incubated in 3% H₂O₂ for 10 min and then with anti-CRABP2 (1:120; Proteintech, Chicago, IL, USA) and anti-FABP5 (1:120; Proteintech, Chicago, IL, USA) at 4 °C for one night in a humid chamber. Finally, the coverslips were co-incubated with FITC-conjugated goat anti-mouse IgG and PE-conjugated goat anti-rabbit IgG (both 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 37 °C for 60 min in the dark, sealed with fluorescence mounting medium, and observed and imaged under a fluorescence microscope (BX53F, Olympus, Tokyo, Japan).

Western blotting was conducted using antibodies against CRABP2 (Proteintech, Chicago, IL, USA; 1:200), FABP5 (Proteintech, Chicago, IL, USA; 1:200), RAR-β (Bioss. Inc., Beijing, China; 1:150), PPAR-β/δ (Bioss. Inc., Beijing, China; 1:200), pro-caspases-3, and active-caspases-3 (Abcam Inc., Cambridge, UK; 1:500 and 1:500). The experiment was performed by the method described elsewhere [15 (link)]. Briefly, the sample proteins (20 µg/well) were separated by 10% SDS-PAGE electrophoresis and transferred to polyvinylidene difluoride membrane (Amersham, Buckin ghamshire, UK). The membrane was blocked in 5% skimmed milk (Sigma-Aldrich) Tris-buffered saline (TBS-T) (10 mM Tris–HCl, pH 8.0, 0.5% Tween 20 and 150 mM NaCl) at 4 °C overnight. After three washes with TBS-T, the membrane was incubated for 3 h with the primary antibody at room temperature, followed by 1 h incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Zymed Lab Inc., San Francisco, CA, USA). The enhanced chemiluminescence system (Roche, Penzberg, Germany) was used to detect the bound antibody. The labeling signal was removed with a stripping buffer (62.5 mM Tris–HCl, pH 6.7, 100 mM 2-mercaptoethanol, 2% sodium dodecyl sulfate (SDS), and the membrane was reprobed with another antibody until all the parameters were examined.