Figure 1.

A TIM4-affinity ELISA screen in U87MG cells identified eight activators and one inhibitor of extracellular vesicles (EVs). (a) The principle of TIM4-ELISA. EVs are captured by immobilized TIM4-Fc proteins via calcium-dependent binding to phosphatidylserine on the surface of EVs. Then, the captured EVs are detected with a primary antibody against an EV surface marker, such as, anti-CD9, anti-CD63, or anti-CD81, and an HRP-conjugated secondary antibody. (b) K562 cells were stimulated for 24 h with 0.1, 1, or 10 μM monensin. EVs contained in the cultured supernatants were detected using TIM4-CD63 ELISA (left) and nanoparticle tracking analysis (NTA; right). (c) In the first round of screening, U87MG cells were treated with a 1,567-compound library at 0.1, 1, or 10 μM for 24 h. EVs contained in the cultured supernatants were detected using TIM4-CD63 ELISA. A typical result is shown here. The two dashed lines represent the threshold values for activators and inhibitors, at 0.67 and 1.5, respectively. (d–m) U87MG cells were treated with 10 μM AA2, 7 μM amlodipine, 2 μM osimertinib, 1 μM cucurbitacin B, 2 μM doramectin, 10 μM gossypol, 15 μM HA14-1, 20 μM miltefosine, or 1 μM obatoclax for 24 h. Cytotoxicity and cell growth were determined using lactate dehydrogenase (LDH) (d, i) and WST-8 (e, j) assays. Cells treated with lysis buffer were used as a positive control (PC) in the LDH assay. Secreted EVs were detected using TIM4-CD9 or TIM4-CD63 ELISA (f, k), Secreted EVs were recovered using ultracentrifugation and subjected to western blot with an anti-CD9 or anti-CD63 antibody (g, l), and NTA (h, m). Full-length blots can be found in the supplementary information. *p < 0.05, **p < 0.01, versus DMSO, Student’s t-test.