Cleavage of HK caused by cancer cell–derived EV. (A) HK cleavage in NHP incubated with cancer cell–derived EV (0.5 and 1 μg/mL) and analyzed by immunoblotting. (B) HK cleavage by EVs from (A) expressed as the percentage of cHK relative to intact HK. (C) HK cleavage 60 minutes after the addition of L3.6 EV following treatment of EV with CIP, DNase I, or RNase A. DS was used as a positive control. (D) Effect of CIP, DNase I, and RNase A on the cleavage of HK by EV from cancer cells and HDFs; the ratio of cHK/HK was determined by immunoblotting and infrared quantification. (E) Effect of CTI on L3.6 EV-induced cHK generation. L3.6 EV were incubated with NHP in the presence or absence of CTI (10 μg/mL) for 60 minutes. For HK cleavage analysis, a polyclonal rabbit anti-human HK antibody (D5; supplemental Figure 1) was used to detect HK and cHK. Bars represent means ± SEM. ***P < .001, 2-way ANOVA with multiple comparisons. LC1, light chain 1 (54 kDa); LC2, light chain 2 (47 kDa). ns, not significant.