Figure 4.

AA2, cucurbitacin B, gossypol, and obatoclax can regulate EV bioactivity in vitro. (a–f) DMSO-, AA2-, CuB-, or Oba-EVs were isolated from the cultured supernatant of U87MG cells following treatment for 2 days with DMSO, AA2, cucurbitacin B, or obatoclax. MG6 cells were stimulated with EVs isolated from the same number of U87MG cells (a, b; 1 × 10⁷ cells, c, d; 3 × 10⁶ cells) or with the same number of EV particles (e; 1 × 10¹⁰ particles, f; 3 × 10⁹ particles) for 12 h. Total RNAs or cellular proteins were extracted from MG6 and the expression of Thbs1 was detected using RTqPCR (a, c, e, f) or western blot (b, d). **p < 0.01, n.s.; not significant, versus DMSO-EV, Student's t-test. A THBS1 band intensity in (b, d) was normalized to β-actin. Full-length blots can be found in the supplementary information. (g) DMSO-, Gos-, or Oba-EVs were isolated from the cultured supernatant of hMSCs following treatment for 2 days with DMSO, gossypol, or obatoclax. H9C2 cells pre-treated with 1 mM CoCl₂ were stimulated with the EVs isolated from 4 × 10⁵ hMSCs for 4 days. Cell growth was measured via a luminescent cell viability assay. **p < 0.01, versus DMSO-EV, Student’s t-test.