Anti cbx4

NP cells were inoculated in a 24-well culture plate and cultured to 70% confluence. Cells were fixed with 4% paraformaldehyde fix solution and permeabilized with 0.5% Triton X-100. Goat serum was used to block the non-specific binding sites before the specimen were incubated with an anti-p65 (1:200; Abclonal), anti-CBX4 (1:200; Abcam), or anti-COL2A1 (1:200; Abcam) antibodies overnight at 4°C, followed by incubation with an Alexa Fluor 488-conjugated anti-rabbit IgG secondary antibody (ZSGB-BIO) for 1 h at room temperature. The cell nuclei were stained with DAPI (Solarbio) or Hoechst (Solarbio). Afterwards, the immunofluorescence signals of proteins were detected with a fluorescence microscope (Olympus).

Human NP tissue was obtained from patients who underwent lumbar interbody fusion for degenerative disc diseases, and the procedures were approved by the Medical Ethics Committee of Sun Yat-sen Memorial Hospital, Sun Yat-sen University. From September 2020 to March 2021, 10 disc samples (male:female=3:7) were collected, evaluated by Pfirrmann classification and classified into 2 groups.
Human tissues paraffin sections were deparaffinized with xylene and rehydrated, followed by retrieval with EDTA buffer (ZSGB-BIO, Beijing, China) for 10 min at 100°C. Hydrogen peroxide (3%) was used to quench endogenous peroxidase activity. Then, goat serum (ZSGB-BIO) was used to block the paraffin sections for 30 min. The paraffin sections were incubated with PBS or anti-CBX4 (1:100; Abcam, Cambridge, UK) primary antibodies overnight at 4°C. After being incubated with biotin-labeled secondary antibodies for 30 min at room temperature and HRP-conjugated streptavidin for 20 min, the sections were developed with DAB solution (ZSGB-BIO), and counterstained with 1% hematoxylin. Lastly, sections were mounted, photographed under a microscope (Nikon, Tokyo, Japan).

Stimulated NP cells were collected and then lysed with RIPA lysis buffer (CWBIO, Beijing, China) for total protein, or with an extraction kit for cytoplasmic and nuclear protein (CWBIO). After being centrifuged and collected, the supernatant containing proteins was then quantified using a BCA assay kit (CWBIO). Afterwards, the proteins were separated by SDS-PAGE and electro-transferred to polyvinylidene difluoride membranes (Millipore, Darmstadt, Germany). After being blocked with 5% bovine serum albumin in TBST for 1 h, the membranes were then incubated at 4°C overnight with anti-CBX4 (1:1000; Abcam), anti-MMP3 (1:1000; Abcam), anti-MMP13 (1:1000; Abcam), anti-ADAMTS5 (1:1000; Abcam), anti-COX2 (1:1000; Abcam), anti-COL2A1 (1:1000; Abcam), anti-P53 (1:1000; Abcam), anti-P21 (1:1000; Abcam), anti-β-actin (1:1000; Abclonal, Wuhan, China), anti-p-p65 (1:1000; Abclonal), anti-p65 (1:1000; Abclonal), or anti-Histone H3 (1:1000; CST, Danvers, USA) antibodies. Afterwards, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies (1:8000; Abclonal). The protein bands were visualized using an Enhanced Chemiluminescence kit (Vazyme, Nanjing, China) and then quantified by Image J (National Institutes of Health, Bethesda, USA).