Fig. 3

Co-culture of H. pylori (Hp), NFs, and gastric cancer cells induces cytokines release to promote NFs activation. A Western blot shows FAP expression in the direct and Transwell (indirect) co-culture of H. pylori, NFs, and AGS cells for the indicated time at a multiplicity of infection (MOI) of 50. The bar graph shows the quantitation of FAP levels. *P < 0.05, **P < 0.01, and ****P < 0.0001 vs. NF. B Western blot shows FAP and Lumican (LUM) expression in the direct and Transwell co-culture of H. pylori, NFs, and AGS or primary GC cells (GC) for 7 days at an MOI of 50. The bar graph shows the quantitation of FAP and LUM levels. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs. NF. C Transwell migration and Matrigel invasion assays in the co-culture of H. pylori, NFs, and AGS cells for 7 days at an MOI of 50. The bar graph shows the quantification of migrated or invaded cells. *P < 0.05 and **P < 0.01 vs. AGS/medium. ^#P < 0.01 vs. AGS/NF. D Cytokine detection in the culture or co-culture supernatants of H. pylori, NFs, and AGS cells for 7 days using a human cytokine array. Black rectangles indicate Reference Spots. Blue rectangles indicate the negative control. The bar graph shows the quantification of cytokines, expressed as the average density of two cytokine spots divided by the average density of six Reference Spots. **P < 0.01 and ****P < 0.0001 vs. NF cells. ^####P < 0.0001 vs. NF + Hp group. E ELISA shows Serpin E1 levels in the culture or co-culture supernatants of H. pylori, NFs, and AGS cells. Data are presented as means ± SD from three independent experiments. **P < 0.01 and ****P < 0.0001. F–H Transwell assays, cytokine detection by the human cytokine array, and Serpin E1 detection by ELISA in Transwell co-culture of H. pylori, CAFs, and AGS cells for 7 days. **P < 0.01, ***P < 0.001, and ****P < 0.0001. ^a P < 0.001 and ^b P < 0.05 vs. AGS/Medium