Fig. 4

Runx1p66 protein expression is short-lived and provides a survival advantage. a Representative Western blot of Runx1 protein expression in seven EV_ARDS and two EV_Ctrl preparations. Normalization was achieved to the volume of blood used (typically 1.5 ml) for EVs isolation. b Time course analysis of Runx1 in EV_ARDS of a long-term ARDS survivor (S6) and panel c, a non-surviving ARDS patient (S12). Expression of Runx1p52 gradually increases from day 1 to day 37 in ICU in the EV_ARDS of long-term survivor. Even if the number of blood samples available for later time points assessed was lower due to death of some patients, the time course analysis data are representative for at least three long-term survivors and three non-surviving ARDS patients. d Western blot of Runx1 expression in lysates of cultured human bone marrow-derived MSCs (d2–d8) as well as in lysates of the EV_MSC released at d7 (d1). 30 μg protein/lane; n = 3. e The Kaplan–Meier statistic shows the improved survival for the EV_ARDSAlk5Runx1p66 patients. Out of 33 ARDS patients included in the study, only 29 patients (multiple samples) were used to investigate the Runx1 isoform content and the Runxp66/p52 ratio. f Four EV_ARDS preparations (p1–p4; isolated from 1.8 ml of blood) were labeled with a biotinylated Alk5 rat Ab followed by streptavidin-conjugated magnetic beads. An isotype matched IgG for Alk5 was used as control. Aliquots of EV_ARDS lysates, unbound and bound fractions were analyzed by SDS PAGE and Western blot with Alk5 and Runx1 Abs. EV_ARDS are immunoreactive to Runx1p52 isoform only as the blood samples used belong to either non-surviving ARDS subjects or to surviving ARDS subjects, but collected in week 1 of ICU stay, when the EV_ARDS do not show Runx1p66 immunoreactivity. n = 9 different ARDS patients; Three independent experiments were performed