A20777

Proteins were extracted from gastric tissues or GES-1 cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China), and protein quantification was conducted using the BCA method (Beyotime, China). Equal amounts of proteins were then separated on a 10% SDS-PAGE gel and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane. Following blocking for 10 min at room temperature using rapid blocking solution (Ncmbio, Suzhou, China), PVDF membranes were incubated with primary antibodies overnight at 4 °C. Afterward, the PVDF membrane was washed three times using tris−buffered saline (TBST) and incubated with secondary antibody for 1 h at room temperature. Finally, emitter-coupled logic (ECL) substrate (Ncmbio, China) was added dropwise, and the membrane was observed using a chemiluminescent detection system (Bio-Rad, Hercules, CA, USA). Quantification of protein bands was performed using Image J software (1.53K). The antibodies used were as follows: β-actin (AB0035, 1:5000, Abways, Beijing, China), Bax (A20227, 1:2000, ABclonal, Wuhan, China), Bcl2 (A20777, 1:1000, ABclonal, China), TNF−α (ER1919-22, 1:500, HuaBio, Hangzhou, China), IL−6 (R1412-2, 1:500, HuaBio, China), and NF−κB (PSH0−27, 1:1000, HuaBio, China).

Whole organoids were collected into 1.5 mL microcentrifuge tubes and centrifuged at 1,500 rpm for 5 min. Organoids were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS and incubated in blocking buffer with 5% BSA for 30 min. Subsequently, primary antibodies to p16 (A11651, 1:100 ABclonal, China) and CCNA2 (A19036, 1:100 ABclonal, China), BCL2 (A20777, 1:100, ABclonal, China) were incubated at 4°C overnight. The secondary antibodies (GB22301 and GB22403, 1:200, Servicebio, China) were incubated at room temperature for 50 min, and then counterstaining with DAPI was performed at room temperature for 10 min in the dark. Images were captured with an inverted fluorescence microscope (TS2R-FL, Nikon, Japan).