EVs promote endosomal trafficking and invasiveness via a TLR9-dependent mechanism. (a and b) Glutamine-starved cells were plated onto dishes coated with fluorescently conjugated gelatin (FITC-gelatin; a) or fibroblast-derived ECM (b) in the presence of ODN 2006 (TLR9 agonist; 1 µM) or ODN 2137 (control [Ctrl] agonist; 1 µM). 16 h following this, gelatin degradation (a) and invasive protrusion length (b) were determined as in Fig. 6, c and d, respectively. Values are mean ± SEM; n = 3 independent experiments. ***, P < 0.001; **, P < 0.002; Mann-Whitney U test. Bar, 20 µm; bar in zoom, 2 µm. (c) Recipient cells were transfected with siRNAs targeting TLR9 (siTLR9) or nontargeting siRNA (siNT). TLR9 knockdown was assessed using qPCR (left panel). Recipient cells were glutamine starved and incubated for 3 d in the absence (−) or presence (+) of EVs from glutamine-replete donor cells. Invasive protrusion length was then determined in the absence or presence of ODN-TTAGGG-A151 (TLR9 antagonist; 1 µM) or ODN-TTAGGG-control (Ctrl antagonist; 1 µM) as in Fig. 6 d. Whiskers are 10th–90th percentiles, + represents the mean. **, P < 0.01; ***, P < 0.001; two-way ANOVA with Dunn’s multiple comparison test. (d) Recipient cells were transfected with stealth siRNAs targeting TLR9 (siTLR9) or nontargeting siRNA (siNT). TLR9 knockdown was assessed using qPCR (left panel). Transfected recipient cells were glutamine-starved, plated onto fluorescently conjugated gelatin, and incubated for 3 d in the absence (−) or presence (+) of EVs from glutamine-replete donor cells. Bar, 20 µm; bar in zoom, 2 µm. The ability of these recipient cells to degrade collagen was then determined as in Fig. 6 d. Values are mean ± SEM; n = 3 independent experiments. **, P < 0.01; ***, P < 0.001; one-way ANOVA with Dunn’s multiple comparison test.