Figure 4

Mitochondrial tracking demonstrated the AEC-derived MV cargo internalization in immune cells

(A) Experimental design for B-D.

(B) Representative confocal images of co-immunofluorescence staining of nuclei (DAPI), MitoTracker green (PBMCs’ mitochondria), and MitoTracker red (MV’ mitochondria) in PBMCs ± EIPA exposed to AEC ± LPS_MV for 12h.

(C) Flow cytometric investigation to confirm the mitochondria MV internalization (MitoTracker red) ± LPS in PBMCs and Jurkat ± CD44 and ± EIPA. Data are presented as % of cells positive to MitoTracker red.

(D) qPCR analysis to corroborate the mitochondria MV internalization after 24h of exposition to AEC ± LPS_MV by examining the ovine mtDNA copy number in Jurkat ± EIPA. (CTR: untreated Jurkat; CTR+: AEC_MV per se). Data (mean ± SD) represent 3 independent sets of experiments (n = at least 3 biological replicates in each group per set; each biological replicate assayed in at least 3 technical replicates). ALL, ∗, ∗∗, ∗∗∗, and ∗∗∗∗ Statistically significant values between the different studied groups (p < 0.0001, p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively). For the transfer of AEC_MV mitochondria into AEC see Figure S1. One-way ANOVA was employed for comparing normally distributed data. Scale bar, 20 μm.