Spin count

Lung tissue harvested 7 days following injury (n = 5, 10, 7, 7, 11 for control, bleo, bleo + CNP, bleo + miR146a, bleo + CNP-miR146a) was homogenized in sucrose buffer in a 1:6 ratio of lung weight to buffer volume (0.25 M sucrose, 10 mM TRIS–HCl, 1 mM EDTA, pH = 7.4). 60 μL of lung homogenate for each sample was mixed with 140 μL of Krebs-HEPES buffer (KHB) consisting of 100 μM DTPA and 0.2 mM CMH. A blank sample of 200 μL KHB was used for background signal subtraction. The samples were incubated for 1 h at 37 °C and 150 μL samples were loaded in PTFE tubing with rubber seal stoppers.³³ ROS production was measured by EPR spectrometry using the superoxide selective spin probe 1-hydroxy-3-methoxycarbonyl-2, 2, 5, 5-tetramethylpyrrolidine (CMH). The Bruker EMX nano X-band spectrometer was used at 77 K with Bruker liquid nitrogen Finger Dewar to detect ROS as CM•. EPR acquisition parameters were: microwave frequency = 9.65 GHz; center field = 3438 G; modulation amplitude = 4.0 G; sweep width = 150 G; microwave power = 0.316 mW; total number of scans = 10; sweep time = 60 s; and time constant = 1.28 ms. CM• nitroxide radical concentration was obtained by double integration followed by Spin count (Bruker).