P16 antibody

10% SDS-PAGE was used to segregate the protein lysates, then incubated together with distinct antibodies, and lastly densitometry (Bio-Rad) was used to quantify the electrophoretic bands. The EZH2 antibody (1:1000) were acquired from Cell Signaling Technology (Cat#: 5246), and the GAPDH served as control (CST, Cat#: 5174). The p16 antibody was also from Cell Signaling Technology (Cat#: 18769).

The following drugs and reagents were used in the study: DMEM/F12 media without phenol red (Gibco), fetal bovine serum (FBS, Sciencell), RNA isolation kit, Color SYBR Green qPCR Master Mix and 4× reverse transcription Mix (EZBioscience, A0012), enzyme linked immunosorbent assay (ELISA) kits (E2, Labor Diagnostika Nord, FR E-2000; FSH, Immunoway, KE1425; LH, Immunoway, KE1475; AMH, Jingmei, JM11692M1; insulin, Alpco, 80-INSMSU-E01, E10), Normal rabbit IgG (CST, 2729), Normal mouse IgG (CST, 7076), p53 antibody (Abcam ab131442), p16 antibody (Cell Signaling Technology Cat, sc1661), p21 antibody (Cell Signaling Technology, sc-6246), AGER antibody (Abcam, ab3611), Senescenceβ-Galactosidase Staining Kit (Beyotime, C0602), Cell Cycle Staining Kit (Multi Sciences, CCS021), insulin (Absin, abs42019847), metformin (Topscience, T8526), high-fat diet (Jiangsu Xietong Pharmaceutical Bio-engineering Co., Ltd., D12451).

The tissue sections were deparaffinized and 3% H₂O₂ was used to block the endogenous peroxidase. The sections were incubated in tris-buffered saline (TBS) with 5% albumin bovine V (BSA; Solarbio, Beijing, China) for 1 h. The cell samples were washed with PBS three times and fixed with 4% paraformaldehyde for 15 min. After washing with PBS three times, tissue sections or cells were permeated with 0.3% Triton X-100 for 15 min and then incubated with p21 antibody (1:200; Abcam, Cambridge, MA, USA), p16 antibody (1:200; Cell Signaling Technology, Danvers, MA, USA), LC3 antibody (1:200; CST, Danvers, MA, USA), PCNA antibody (1:200; Proteintech, Wuhan, China) or Ki67 antibody (1:200; Abways, Shanghai, China) in 5% BSA overnight at 4 °C. After washing with TBS, the sections were incubated with a Rhodamine (TRITC)-conjugated goat anti-rabbit IgG (H+L) (1:300; Abcam, Cambridge, UK). The nuclei were counterstained with DAPI (Invitrogen, Waltham, MA, USA). The sections were washed with PBS three times, and coverslips were mounted in 90% glycerol in PBS. The fluorescence was detected by a fluorescence microscope (Nikon, Tokyo, Japan). Image J was used to analyze the fluorescence intensity.