Gastric cancer cell-derived conditioned medium induced autophagy and pro-tumor activation of neutrophils. a. Flow cytometric analyses for apoptosis in neutrophils treated with or without conditioned medium from BGC-823 gastric cancer cells (BGC-CM). b. The expression of CD11b in BGC-CM-treated neutrophils was determined by flow cytometric analysis. c. Transwell migration assays for gastric cancer cells following treatment with supernatant from BGC-CM-treated neutrophils. d. Transmission electron microscopy analyses of autophagosomes (arrows) in neutrophils treated with BGC-CM. Scale bar=1 μm. e. Immunofluorescent staining of LC3-positive puncta in neutrophils treated with or without BGC-CM. Nuclei were counterstained by Hoechst 33342 (blue). f. Western blot assays for the expression of LC3-II in neutrophils incubated with conditioned medium from gastric cancer cells. g. The expression of ATG7 and BECN1 genes in neutrophils treated with conditioned medium from gastric cancer cells was determined by qRT-PCR. h. Neutrophils were pre-treated with autophagy inhibitors 3-MA or CQ followed by incubation with BGC-CM. The percentage of apoptotic neutrophils was determined by using flow cytometry. i. FACS analyses of CD11b expression in neutrophils treated with 3-MA and CQ prior to exposure to BGC-CM. j. Gastric cancer cells were incubated with the supernatants from BGC-CM-treated neutrophils that were pre-treated with or without autophagy inhibitors 3-MA or CQ and used for transwell migration assay. **P<0.01 and *P<0.05 compared to control (Ctrl); ##P<0.01 and #P<0.05 compared to BGC-CM.