Figure 2
AA2 inhibited EV secretion independent of nSMase2 and caspase 3. (a, b) U87MG cells were treated for 24 h with #1, #7, or #10 at 1 μM; AA2, #2–#6, #8, or #9 at 10 μM, and then secreted EVs were determined using TIM4-CD9 or TIM4-CD63 ELISA (a) or NTA (b). (c, d) Cell lines were treated with AA2 as shown in Table 3 (c) or 3 μM GW4869 (d) for 24 h and then secreted EVs were determined using TIM4-ELISA. EVs secreted from THP-1, Jurkat, and EL4 cells were detected using TIM4-CD81 ELISA. EVs secreted from SW620, U87MG, SW480, HEK293T, HCT116, and NIH/3T3 cells were detected using TIM4-CD63 ELISA. (e–g) U87MG cells were treated with 30 or 60 μM MT-21 for 24 h. Cytotoxicity and cell growth were determined using LDH (e) and WST-8 (f) assays. Secreted EVs were determined using TIM4-CD9 or TIM4-CD63 ELISA (g). (h–k) Jurkat cells were pre-treated with 0 or 50 μM Q-VD for 3 h, and then with 0 or 5 μM AA2 for 24 h. Cytotoxicity and cell growth were determined using LDH (h) and WST-8 (i) assays. (j) The cells were lysed and immunoblotted with anti-caspase 3, anti-cleaved-caspase 3, or anti-β-actin antibody. Full-length blots can be found in the supplementary information. (k) Secreted EVs were determined using TIM4-CD81 ELISA. (l) HEK293T WT or CASP3 KD cells were lysed and immunoblotted with anti-caspase 3 or anti-β-actin antibody. Full-length blots can be found in the supplementary information. (m, n) HEK293T WT or CASP3 KD cells were treated with 3 μM AA2 for 24 h. Cell growth was determined using a WST-8 assay (m) and secreted EVs were determined in TIM4-CD81 ELISA (n). *p < 0.05, **p < 0.01, n.s.; not significant, versus DMSO or AA2-treated WT, Student’s t-test.
