Anti cyclin d1

For immunofluorescence analysis, SMSCs were seeded onto 24-well plates at 1 × 10⁴ cells per well and then treated with tFNAs (0, 250 nM). After 24 h of treatment, the cell samples were rinsed with PBS three times and fixed in cold 4% formaldehyde solution for 20 min. After 3 washes with PBS again, Triton X-100 (0.5%) was used to permeabilize the SMSCs for 30 min, and then, the samples were blocked with immune blocking solution (Beyotime, Shanghai, China) for 30 min. Fixed SMSCs that were rinsed with PBS were then incubated with anti-β-catenin (1:200, Abcam, Cambridge, England), anti-Lef-1 (1:200, Novus, NY, USA), anti-cyclin D1 (1:200, Novus, NY, USA), anti-phospho-Smad2 (1:2000, Cell Signaling Technology, MA, USA), and anti-phospho-Smad3 (1:200, Cell Signaling Technology, MA, USA) primary antibodies overnight at 4 °C. Fluorescence-conjugated secondary antibody was then combined with primary antibodies for 1 h at 37 °C. DAPI (1:200; Beyotime, Shanghai, China) was applied to stain the cell nucleus for 10 min, and phalloidin (1:60; Beyotime, Shanghai, China) was used to stain F-actin for 40 min. Finally, cell images were observed by fluorescence microscopy (Keyence, Osaka, Japan).