Tnfaip2

Formalin-fixed paraffin-embedded U87 intracranial tumors were cut into 6-μm-thick sections on a microtome. Immunohistochemical staining was performed according to standard procedures. Antigen retrieval was conducted in 0.1 M citrate buffer (pH 6.0) at 95°C for 16 min and cooled at 25°C for 1 h. After blocking, slides were incubated with primary antibodies TNFAIP2 (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), SOX2 (1:200, Boster Bioengineering Co., Wuhan, China) and ki-67 (1:200, Boster Bioengineering Co., Wuhan, China). Next, according to the manufacturer’s instructions, slides were treated with the Cell & Tissue Staining Kit HRP-DAB system (R&D Systems, Minneapolis, MN, USA). The immunohistochemical staining results were evaluated by two experienced pathologists.

Total protein in NCM460 was extracted after treatment of TNF-α and IFN-γ for 48 h. The protein in cells and colonic mucosa was extracted using RIPA lysis buffer (Boyotime, China) according to the reagent instructions. Western blotting was performed with the specific antibody, STAT1 (1:1000, Cell Signaling Technology, 14994S), p-STAT1 (1:1000, Cell Signaling Technology, 9167S), GAPDH (1:1000, Proteintech, 60004-1-Ig), EP300 (1:1000, Cell Signaling Technology, 4771 T), H3K27ac (1:1000, Cell Signaling Technology, 8173S), TNFAIP2 (1:500, Santa Cruz, sc28318), and LCP2 (1:1000, Santa Cruz, sc-13151).