FIG 3.

RV stimulates NLRX-1-dependent mitochondrial ROS in polarized airway epithelial cells. The polarized monolayers of 16HBE14o− cells were infected with RV or sham control and incubated for 90 min at 33°C. The infection medium was replaced with fresh medium, and incubation continued for an additional 16 h. The cells were washed once with HBSS and incubated with 5 μM MitoSox Red for 15 min at 37°C. The cells were washed with warm HBSS. (A) Cells were incubated with HBSS containing Hoechst dye (nuclear stain) for 10 min, which was then replaced with warm HBSS, and the cells were imaged immediately. The images are representative of 3 independent experiments (red, MitoSox Red fluorescence indicative of mitochondrial ROS; blue, nuclei). (B and C) Cells were detached from the plate, suspended in warm HBSS, and analyzed by flow cytometry. MFI, mean fluorescence intensity. (D) NT or NLRX-1 siRNA-transfected cells were infected with sham control or RV, and mitochondrial ROS was quantified by flow cytometry 16 h postinfection. (B) Representative histogram of 3 independent experiments. (C and D) Data represent means and SD calculated from 3 or 4 independent experiments (*, P ≤ 0.05; ANOVA).