Fig 2: Effect of DJ-1 and DJ-1?C ischemic exposure in signalling and in vitro angiogenesis. ECs cultures were exposed to either 1 h ischemia or normoxia, in the presence and the absence of exogenously administrated DJ-1 or DJ-1?C at 100 nM, with and without a reperfusion period of 2 h, and both the phosphorylation status of Akt and the cleavage of Casp-3 were analysed upon cell lysates. (A) Representative western blot of phospho-Akt (Ser473) and total Akt in normoxia, ischemia, and I/R, in the presence and the absence of full-length DJ-1 or DJ-1?C. (B) Akt relative phosphorylation status quantification (n = 4; **p < 0.01, n.s. not significant). (C) Representative western blot of Casp-3 in normoxia, ischemia, and I/R, in the presence and the absence of full-length DJ-1 or DJ-1?C. No cleavage of Casp-3 was detectable in any condition, an overexposed acquisition of the 17 kDa surrounding region is included. (D) Representative acquisitions of tube-formation assay of ECs during reperfusion after the exposure to 1 h of ischemia, in the presence and the absence of exogenously administrated DJ-1 or DJ-1?C at 100 nM. No exogenous DJ-1/DJ-1?C were added in the reperfusion media. (E) Total tubes length quantification over reperfusion time (n = 3; *p < 0.05). Casp-3 Caspase-3, ECs endothelial cells, I/R ischemia/reperfusion. Corresponding uncropped western blot acquisitions can be found in Supplemental Figs. 6 and 7.

Fig 3: Systemic DJ-1 administration effects on oxidative stress after ischemia and ischemia/reperfusion. (A) Inos transcript analysis by qPCR. (B) Representative immunohistochemical acquisitions of myocardial iNOS (upper panel) and 8-OHdG (lower panel) after ischemia and ischemia/reperfusion in the presence and the absence of a systemic administration of DJ-1. A sham-operated group was included as baseline. (C) Myocardial iNOS content quantification. (D) Myocardial 8-OHdG content quantification. (E–G) Correlation analyses between iNOS and DJ-1 (E), 8-OHdG and DJ-1 (F), and iNOS and 8-OHdG (G) staining signals. Scale bar: 100 µm; *p < 0.05, Kruskal-Wallis; §p < 0.05 vs. sham, †p < 0.05 vs. ischemia without DJ-1, ‡p < 0.05 vs. ischemia + DJ-1, #p < 0.05 vs. I/R without DJ-1, Dunn’s test; rho, Spearman rank correlation coefficient. Isch., Ischemia; I/R, Ischemia/Reperfusion.

Fig 4: ATP-synthase co-immunoprecipitation with exogenous DJ-1 and DJ-1?C in ischemia and I/R. ECs cultures were exposed to either 1 h ischemia or normoxia, in the presence and the absence of exogenously administrated DJ-1 or DJ-1?C at 100 nM, with and without a reperfusion period of 2 h and 24 h (whithout exogenous DJ-1/DJ-1?C). After indicated treatment, cells were thoroughly rinsed, lysed, and immunoprecipitated against ATP-synthase. (A) Western blot analysis of ATP synthase (ATP5B) and DJ-1/DJ-1?C in whole cell lysates (immunoprecipitation input). (B) Western blot analysis of ATP synthase (ATP5B) and DJ-1/DJ-1?C in the IP-captures. ECs endothelial cells, IP immunoprecipitate, I/R ischemia/reperfusion. Membranes were split in two before incubation. Corresponding uncropped western blot acquisitions can be found in Supplemental Fig. 5.

Fig 5: Effects of DJ-1 systemic administration on myocardial leukocyte infiltration after ischemia and ischemia/reperfusion. (A). Representative immunohistochemical acquisitions of myocardial neutrophils (upper panel) and macrophages (lower panel) after ischemia and ischemia/reperfusion in the presence and the absence of a systemic administration of DJ-1. A sham-operated group was included as baseline. (B) and (C) quantification of infiltrated leukocytes. Scale bar: 100 µm; *p < 0.05, Kruskal-Wallis; §p < 0.05 vs. sham, †p < 0.05 vs. ischemia without DJ-1, ‡p < 0.05 vs. ischemia + DJ-1, #p < 0.05 vs. I/R without DJ-1, Dunn’s test. Isch., Ischemia; I/R, Ischemia/Reperfusion.