Fig. 6: Caspase-3 regulates the release of apoptotic exosome-like vesicles (ApoExos).

A Hoechst 33342 and propidium iodide (HO/PI) staining of apoptosis serum-starved (SS) HUVECs exposed to vehicle (V) or DEVD as well as wild-type (WT) or caspase-3 knockout (Casp3 KO) murine endothelial cells (mECs). P values were obtained by the unpaired t test. n = 3 for V or DEVD treatments, and n = 7 for WT and Casp3 KO. B Representative immunoblots and densitometric analysis of LG3, 20S proteasome, and LAMP2 in ApoExos purified from HUVECs serum starved (SS) for 4 h and exposed to V or DEVD as well as in ApoExos derived from Casp3−/− and wild-type mECs serum starved (SS) for 9 h. Ponceau red was used as a loading control. P values were obtained by the unpaired t test. n = 4 for each condition. C Representative electron micrograph of WT or Casp3−/− mECs serum-starved (SS) for 9 h showing autolysosomes (AL, representative of two independent experiments). Scale bar = 500 nm. D Quantification of the cell surface occupied by lysosomes, autophagosomes, multivesicular bodies, and autolysosomes in WT or Casp3−/− mECs SS for 9 h. P values were obtained by the unpaired t test. n = 50 cell profiles from two independent experiments for each condition. ns (nonsignificant). E Quantification of the distance between the autolysosomes and the plasma membrane in WT or Casp3−/− mECs SS for 9 h. P values were obtained by the Mann–Whitney test. n = 50 cell profiles from two independent experiments for each condition. *P < 0.05; **P < 0.01; ***P < 0.001. All values are expressed as the mean ± SEM.