Fig. 1. NDUFS8 silencing impairs mitochondrial functions in cultured endothelial cells.
NDUFS1/NDUFS8 expression in stable HUVECs treated with NDUFS8 shRNA (“kdNDUFS8-sh2” and “kdNDUFS8-sh5”) or the scramble non-sense shRNA (“kdC”) was shown (A, B). Following a 24 h culture, the mitochondrial respiratory chain Complex I activity (C), cellular ATP levels (D), reduction in mitochondrial membrane potential (tested via mitochondrial JC-1 staining, E), ROS contents (measured using MitoSOX dye, F), and lipid peroxidation (via BODIPY staining, G) were shown. Similarly, human microvascular endothelial cells (hRMEC), human dermal endothelial cells (hDEC), and human cerebral microvascular endothelial cells (hCMEC) with either kdNDUFS8-sh5 or kdC were established. NDUFS8 (H) and NDUFS1 (I) mRNA expression was quantified, followed by a 24 h culture and examination of mitochondrial respiratory chain Complex I activity (J), cellular ATP content (K), mitochondrial depolarization (by measuring JC-1 green monomers intensity, L), and ROS production (by measuring MitoSOX intensity, M). “Pare” denotes the parental control endothelial cells. The data are presented as mean ± standard deviation (SD, n = 5). *P < 0.05 compared to “Pare”/“kdC” cells. “N. S.” represents non-statistically significant disparities (P > 0.05). These experiments were repeated five times, yielding consistent results. Scale bar = 100 μm.