Fig 2: p65 directly binds to the INHBB promoter and induces INHBB expression in GC cells. (A) RT-PCR analysis was conducted to determine the expression levels of INHBB mRNA in cells treated with IL-1b. (B) Western blot analysis was conducted to determine the expression levels of INHBB protein in cells treated with IL-1b. C-D. Luciferase reporter vectors containing wild-type or mutants INHBB and p65 or the vector were transfected into HGC-27 and MGC-803. Luciferase activities were measured using luciferase assays. E-F. The wild type and mutants INHBB luciferase reporter vectors were transfected into HGC-27 and MGC-803 cells. Treatment with IL-1b or transfection with p65 or pcDNA3.1, the corresponding relative luciferase activity was determined by luciferase assays (n = 3). G. p65 binding motif. H. Jasper transcription factor binding site prediction supplied two potential p65-binding sites of INHBB. I. Agarose electrophoresis performed to verify p65 binding to the INHBB promoter. J. Correlation analysis of mRNA levels between INHBB and p65 based on TCGA database using GEPIA. K. Representative IHC staining images of INHBB and p65 in GC tissues (n = 90). L. The correlation between INHBB and p-p65 levels in 90 GC tissues was analyzed. ***, P < 0.001; ****, P < 0.0001. p65: RELA.

Fig 3: Activated fibroblasts regulate GC cell function via the IL-1β/p65 pathway. (A) A Venn diagram summarized the up-regulated cytokine genes in fibroblasts stimulated by exogenous activin B or co-cultured with GC cells (HGC-27 shNC vs. HGC-27 shINHBB; MGC-803 vs. MGC-803 shINHBB; MKN-45 INHBB vs. MKN-45 Vector). (B) mRNA levels of IL-1b was examined by qRT-PCR in GC cells (HGC-27 shNC/HGC-27 shINHBB, MGC-803/MGC-803 shINHBB, MKN-45 Vector/MKN-45 INHBB, HGC-27 Vector/HGC-27 INHBB). (C) Western blot analyses of the levels of p-p65 and p-65 in GC cells co-cultured with fibroblasts. (D) Western blot analyses of the levels of p-p65 and p-65 in GC cells treated with IL-1b for different times. (E) Cell proliferation was verified by CCK-8 assay. The value of the absorbance (at 450 nm) was recorded from 0 to 96 h in GC cells treated or untreated with IL-1b (n = 3). (F) Cell migration and invasion were detected by transwell assay in GC cells with or without IL-1b co-culture (n = 3). (G) Western blot analyses of the levels of p-p65 and p-65 in GC cells treated and untreated with JSH-23 in fibroblasts co-cultured system. (H) Cell proliferation was verified by CCK-8 assay. The value of the absorbance (at 450 nm) was recorded from 0 to 96 h in GC cells treated or untreated with JSH-23 in fibroblasts co-cultured system (n = 3). (I) Cell migration and invasion were detected by transwell assay in GC cells in fibroblasts co-cultured system with or without JSH-23 co-culture (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. rActivin B: Recombinant Human Activin B; Sup.: supernatant