Hyperoxia stimulates MLE-12 cells to release EVs with an increased cargo of GSDMD-p30 that induce NSC death in vitro. MLE-12 cells were cultured under RA or 95% O2 for 48 h. EVs in the culture media were isolated and analyzed by nanosight tracking, (A) EVs from RA-maintained MLE-12 cells (RA-MEVs) and (B) EVs from O2-exposed MLE-12 cells (O2-MEVs). (C) Representative western blots of GSDMD, SPC and CD9 (Supplemental Fig. 7C). O2-MEVs had decreased GSDMD-p53 cargos ((D), **P < 0.01) and increased GSDMD-p30 cargos ((E), ***P < 0.001) compared to RA- MEVs. n = 3/group. NSC were cultured in the presence of RA-MEVs (F, H, K) or O2-MEVs (G, I, L) for 48 h. (F) and (G): live cell imaging under phase contrast showed blebbed cells (black arrow) in O2-MEVs treatment. (H) and (I): TUNEL (green signals) and DAPI nuclear staining (blue signals) showed increased cell death in O2-MEVs treated NSC compared to RA-MEVs treated NSC ((J) n = 4/group, ***P < 0.001). (K) and (L): PI uptake (red signals) demonstrated increased pyroptotic cell death with O2-MEVs treatment compared to RA-MEVs treatment ((M) n = 4/group, ***P < 0.001). Scale bar: 50 µm.