Jsh 23

The human GC cell lines MGC803, BGC823, MKN-45, SGC7901, AGS, and the immortalized gastric epithelium cell line (GES-1) were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Primary GC cell line XN0422 was established in our laboratory^{55 (link)}. Cell culture was conducted as previously described^{56 (link)}. The cells were treated as follows: PI3K inhibitor LY294002 (CST) 10 μM for 24 h; AKT inhibitor MK2206 (Selleck Chemicals) 5 μM for 24 h; NF-κB inhibitor JSH-23 (Abcam) 10 μM for 24 h; IKK inhibitor BAY 11–7082 (Abcam) 10 μM for 12 h; NF-κB activator TNF-α (Sigma-Aldrich, USA) 10 ng/mL for 24 h.

HNECs were seeded onto 6-well dishes coated with collagen type I (Kurabo, Osaka, Japan) at 0.6 × 10⁶ cells/well for 24 hours prior to subsequent analysis. The cells were washed with phosphate-buffered saline (PBS), followed by the addition of TLR agonists (Invivogen, San Diego, CA, USA) for 24 hours, unless otherwise described. The following dosages were used: TLR1/2 (Pam3CSK4) 1 µg/ml, TLR2 (HKLM) 10⁸ cells/ml, TLR3 (Poly(I:C)) 10 µg/ml, TLR4 (LPS) 10 µg/ml, TLR5 (Flagellin) 10 µg/ml, TLR6/2 (FSL-1) 1 µg/ml, TLR7 (Imiquimod) 10 µg/ml, TLR8 (ssRNA40) 10 µg/ml, and TLR9 (ODN2006) 5 µM. In some experiments, fluticasone propionate (FP; Cayman Chemical Company, Ann Arbor, MI, USA) and JSH-23 (NFκB transcriptional activity inhibitor; Abcam, Cambridge, MA, USA), was added to the cells, 1 hour prior to TLR stimulation. The following dosages were used: FP 10 nM and JSH-23 30 µM, unless otherwise described. FP and JSH-23 were dissolved in dimethyl sulfoxide (DMSO). In experiments where FP or JSH-23 was used, corresponding DMSO control solutions were also used in parallel as controls.