Ptgs2

Dulbecco’s modified eagle medium (Art. No.10–013-CVRC, Corning, New York, NY, USA); β-sitosterol (Batch No. Y22A10C85758, Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China), fetal bovine serum (Art. No.04–007-1a, Biological Industries, Kibbutz Beit Haemek, Israel), phosphate buffer saline (PBS; Art.No. WH0112201 911 XP, Procell, Wuhan, China), pancreatin (Art. No.143188, Biosharp, Hefei, China), DMSO (Tianjin Fuyu Fine Chemical Co., Ltd., Wuching District, China), MTT (Art. No. M8180, Beijing Suleibao Technology Co., Ltd., Beijing, China), a BCA kit (Lot No.20210922, Bio-Swamp Life Science Lab, Wuhan, China), an ECL high-sensitivity chemiluminescent solution kit (Batch No.: GC 10AA0033, Biological Engineering Co., Ltd., Shanghai, China), β-actin (Batch No.: F200040, Abways Technology, Shanghai, China), VEGFA (Batch No. 83 m8093, Affinity Biosciences, Beijing, China), PTGS2 (Batch No. 86F4760, Affinity Biosciences), VEGFR2 (Batch No. 84 g5912, Affinity Biosciences), and horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody (Batch No. F300405, Abways Technology) were used in this study.

The third-generation human NP cells was tested, remove the culture medium, wash twice with PBS buffer, incubate with 4% neutral paraformaldehyde fixative solution for 5 min at room temperature; wash twice with PBS buffer, 0.1% TritonX-100 cells were incubated with permeabilization solution at room temperature for 20 min; goat serum was blocked for 1 h; blocking solution was removed, Collagen II(1:200), Agreecan (1:200), ERBB2(1:150), PTGS2(1:200),and P16(1:200) (Affinity Biosciences, China) were added, and incubated overnight at 4 °C; primary antibody was removed and washed with PBS for 5 min×3 times, add fluorescent secondary antibody working solution and incubate for 1 h; discard the secondary antibody, wash with PBS for 5 min×3 times, add DAPI staining working solution and incubate for 20 min; observe under an inverted fluorescence microscope and take pictures for recording.

NP cells were lysed in radioimmunoprecipitation assay buffer. Cell lysates were centrifuged at 13,000 rpm for 10 min at 4 °C and the supernatant was collected. Protein samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and were then transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking with 5% nonfat dry milk, the membrane was exposed to the primary antibody (ERBB2 (1:800), PTGS2 (1:800), P16 (1:1000), and P21 (1:1000) (Affinity Biosciences, China) and β-actin (1;5000) (ZSGB-Bio)) incubated at 4 °C overnight. Then membranes were incubated with the appropriate secondary antibodies for 1 h at room temperature. Protein bands were detected using an enhanced chemiluminescence kit and ImageJ software was used to process band intensities.