FIG. 4.

RISP KD blocked caffeine- and hypoxia-induced ROS generation in PASMCs and Ca²⁺-evoked ROS generation in mitochondria. (A) Western blot image of RISP expression in PASMCs uninfected, infected with lentiviral RISP shRNAs, and infected with lentiviral NS shRNAs. (B) Summary of Western blot analysis of RISP expression levels in PASMCs uninfected, infected with lentiviral RISP shRNAs, and infected with lentiviral NS shRNAs. (C) Caffeine (20 mM)-evoked ROS generation, determined by assessing DCF-derived fluorescence, in PASMCs uninfected, infected with lentiviral RISP shRNAs, and infected with lentiviral NS shRNAs. (D) ROS generation (DCF-derived fluorescence) in isolated mitochondria from PASMCs uninfected and untreated with caffeine as well as uninfected, infected with lentiviral RISP shRNAs, and infected with lentiviral NS shRNAs followed by treatment with caffeine (20 mM) for 5 min. (E) ROS generation in isolated mitochondria from PASMCs uninfected and untreated with exogenous Ca²⁺, as well as uninfected, infected with lentiviral RISP shRNAs, and infected with lentiviral NS shRNAs followed by treatment with Ca²⁺ (3 μM) for 5 min. (F) ROS generation in complex III from PASMCs uninfected and untreated with Ca²⁺, as well as uninfected, infected with lentiviral RISP shRNAs, and infected with lentiviral NS shRNAs followed by treatment with Ca²⁺ (3 μM) for 5 min. (G) ROS generation in PASMCs uninfected, infected with lentiviral RISP shRNAs, and infected with lentiviral NS shRNAs followed by normoxic or hypoxic exposure for 5 min. (H) ROS generation in isolated mitochondria from PASMCs uninfected, infected with lentiviral RISP shRNAs, and infected with lentiviral NS shRNAs followed by normoxic or hypoxic exposure for 5 min. (I) ROS generation in MASMC following normoxic or hypoxic exposure for 5 min. (J) Western blot analysis of RISP expression in PASMCs and MASMCs. All data were obtained from at least three separate experiments. *p < 0.05 compared with control; ^#p < 0.05 compared with caffeine- or Ca²⁺-treated samples. KD, knockdown; NS, nonsilencing; RISP, Rieske iron–sulfur protein; shRNA, short hairpin RNA.