Bicinchoninic acid kit

Pre‐lysed in radio‐immunoprecipitation assay cell lysis buffer (Gibco), proteins from cells and tissues were extracted by centrifugation and detected by bicinchoninic acid kit (Sangon Biotech Co., Ltd., Shanghai, China). Successively separated on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes, the protein samples were blocked with 5% skim milk, reacted with primary antibodies HOXA1 (1:1000, ab230513, Abcam, Cambridge, UK), LC3‐II and LC3‐I (1:1000, ABC929, Sigma‐Aldrich, CA, USA) and GAPDH (1:2500, ab9485, Abcam). Next, the protein samples were incubated with secondary antibody (1:1000, ab150077, Abcam) and developed by enhanced chemiluminescence (Millipore, MA, USA) to detect gene protein expression.¹⁴

Total protein was extracted using radio-immunoprecipitation assay (RIPA) lysis buffer (Gibco, Carlsbad, CA, USA) containing phenylmethanesulfonyl fluoride (PMSF), and tested by a bicinchoninic acid kit (Sangon, Shanghai, China). Separated through sodium dodecyl sulphate polyacrylamide gel electrophoresis, the protein samples were electroblotted to polyvinylidene fluoride membranes and blocked with 5% skim milk. After that, membranes were probed with anti-YY1 (1:1000; Santa Cruz Biotechnology, CA, USA) and SYPL1 (1:1000; Abcam, MA, USA) overnight, and with the corresponding secondary antibody for 2 h. Protein bands were developed by enhanced chemiluminescence (Millipore, MA, USA).

Total protein extraction was implemented using radio-immunoprecipitation assay (R0010, Solarbio) containing phenylmethylsulfonyl fluoride. The protein concentration was detected by bicinchoninic acid kit (C503021-0500, Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai, China). Next, 50 μg of protein was separated with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and electrotransferred onto a polyvinylidene fluoride membrane (Merck Millipore, USA). Membrane blockade was conducted using 5% BSA on a shaking table for 1 h, and then the membrane was incubated overnight at 4 °C with the following primary antibodies: rabbit anti-PER2 (ab179813, 1:5000), Runx2 (ab23981, 1:1000), OCN (ab93876, 1:500), β-catenin (ab32572, 1:5000), C-Myc (ab32072, 1:1000), Cyclin D1 (ab16663, 1:4000), Wnt7b (ab94915, 1:1000), GAPDH (ab8245, 1:5000, internal reference), and histone H3 (ab1791, 1:1000). All antibodies were provided by Abcam. Subsequently, the membrane was incubated with the horseradish peroxidase-labeled goat anti-rabbit secondary antibody immunoglobulin G (IgG) (ab6721, 1:5000, Abcam) for 1 h. Following three TBST washes (15 min per wash), the membrane was added with the luminescent solution. The results were analyzed by Bio-Rad gel imaging analysis system (Bio-Rad, Hercules, CA, USA) and Image J.