Figure 3.
MitoKATP participates in ROS generation in hPASMCs under hypoxic conditions via the miR-210/ISCU pathway. The (A) ΔΨm and (B) intracellular ROS levels were measured in hPASMCs transfected with miR-210 mimic or inhibitor for 24 h, prior to normoxia or hypoxia for an additional 24 h. Data are presented as a percentage of control (n=3), and analyzed by a Student's t-test. (C) Expression of ISCU was assessed using western blot analysis upon hypoxia or normoxia, or cells were transfected with miR-210 mimic or inhibitor upon normoxia for 48 h. The β-actin or GAPDH acted as loading control. (D) hPASMCs were transfected with 5 nM of siRNAs against ISCU or si-Con for 48 h. The silencing efficiencies of ISCU (si-ISCU) were measured by western blot analysis. GAPDH acted as loading control. (E) ΔΨm in hPASMCs were transfected with si-Con or si-ISCU upon normoxia for 24 h. The ΔΨm was analyzed by R-123 fluorescence. Data is expressed as a percentage of the control (n=3), and were analyzed by a Student's t-test. *P<0.05, **P<0.01 and ***P<0.001 vs. control. MitoKATP, mitochondrial ATP-sensitive potassium channel; ROS, reactive oxygen species; hPASMCs, human pulmonary artery smooth muscle cells; miR, microRNA; si-Con, scramble control; si, small interfering; ISCU, iron-sulfur cluster protein; ΔΨm, mitochondrial membrane potential; YC-1, 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole; DCFH-DA, 2′,7′-dichlorofluorescin diacetate; R-123, Rhodamine-123; GAPDH, glyceraldehyde 3-phsphate dehydrogenase; con, control; si, small interfering RNA; FITC, fluorescein isothiocyanate.