Anti p53

Total proteins were isolated from cells or tissues using the RIPA lysis buffer (Beyotime, China) and subjected to 10% polyacrylamide gel electrophoresis. After transferring to PVDF membrane, blocking with 5% skim milk, incubation with primary antibodies and secondary antibodies, the proteins on the membrane were finally detected by ECL kit (EpiZyme, China) and imaging analysis system (BioRad, USA). The primary antibodies used in this study were as follows: anti-GAPDH (Bioworld, 1:10000), anti-CDK2 (Bioworld, 1:1000), anti-Cyclin E1 (Bioworld, 1:1000), anti-CDK4 (Bioworld, 1:1000), anti-Cyclin D1 (Bioworld, 1:1000), anti-p53 (Bioss, 1:500), anti-p21 (Bioworld, 1:500), anti-p16 (Bioss, 1:1000), anti-ubiquitin (Santa, 1:500), anti-FTO (Bioss, 1:1000), anti-YTHDF2 (Abcam, 1:1000).

Saw palmetto extract was purchased from Yongyuan Bio-technology, Co., Ltd. (Xi’an, China). Rabbit anti-B-cell lymphoma-extra large (Bcl-xL), anti-p53, anti-PI3K and anti-β-actin antibodies were purchased from Bioss, Inc. (Wuhan, China). An enhanced chemiluminescence (ECL) kit was obtained from the Beyotime Institute of Biotechnology (Shanghai, China).

The paraffin blocks containing the skin samples were sectioned into 5 μm slices and heat-treated for 2 h at 65 °C. A citrate buffer (pH 6.0) was used for antigen retrieval at 121 °C for 2 min. After a blocking process with serum and hydrogen peroxide, the slices were incubated overnight at 4 °C with the anti-p21 (1:1000, Servicebio, Wuhan, China) and anti-p53 (1:200, Bioss, Beijing, China) primary antibodies. Next, the slices were incubated with a horseradish enzyme-labeled goat anti-rabbit immunoglobulin G antibody (1:200, Servicebio, Wuhan, China) at 37 °C for 30 min, stained with hematoxylin and 3,3′-diaminobenzidine. The stained slices were examined using an optical microscope (NIKON ECLIPSE E100, Nikon, Tokyo, Japan), and images were captured using a camera (NIKON DS-U3, Nikon, Tokyo, Japan). Finally, Image J software version 1.53 was employed to determine the positive area.