Fig. 3

Gastric cancer cell-derived exosomes induced autophagy to promote pro-tumor activation of neutrophils. a. The morphology (left) and size (right) of gastric cancer cell-derived exosomes were determined by TEM and NTA, respectively. b. The expression of exosomal markers CD9 and CD63 was determined by western blot. c. The internalization of CM-Dil-labelled exosomes (red) by neutrophils was determined by Imaging flow cytometry. Nuclei were counterstained with Hoechst 33342 (blue). BF, bright field. d. The presence of autophagosomes (arrows) in neutrophils treated with gastric cancer cell-derived exosomes (BGC-Ex) was determined by TEM. Scale bar=1 μm. e. Immunofluorescent staining of LC3-positive puncta (green) in BGC-Ex-treated neutrophils. f. The expression of LC3-II in BGC-Ex-treated neutrophils was confirmed by western blot. g. QRT-PCR analyses of ATG7 and BECN1 expression in BGC-Ex-treated neutrophils. h-j. Neutrophils were pre-treated with autophagy inhibitors 3-MA and CQ followed by treatment with BGC-Ex. FACS analyses of spontaneous apoptosis in neutrophil were shown (h). CD11b expression in neutrophils was determined by FACS analyses (i). Gastric cancer cells were incubated with the supernatants from neutrophils and used for transwell migration assay (j). ^**P<0.01 and ^*P<0.05 compared to control (Ctrl); ^##P<0.01 and ^#P<0.05 compared to BGC-Ex