Fig. 5
GTS-21 inhibits hyperoxia-induced NF-κB activation. a RAW 264.7 cells were exposed either to 95% O2 alone or 95% O2 in the presence of GTS-21 (50 µM) or remained at 21% O2 for 24 h. Following oxygen exposure, cells were washed with PBS, fixed, permeabilized and stained to localize the NF-κB p65 subunit (Red). Multiple pictures were taken using immunofluorescence microscope to visualize the localization of p65 subunit of NF-κB. Counterstaining with DAPI was used to visualize nuclei (Blue). b Immunofluorescent micrographs were quantified to determine the amount of p65 translocation into the nucleus by determining their Mander’s correlation coefficient. The immunofluorescence images shown are representative of two independent experiments. c C57BL/6 mice were exposed to ≥ 99% O2 for 48 h and then inoculated with PA (0.1 × 108 CFUs/mouse), and returned to 21% O2 after inoculation. Mice were randomized to receive either GTS-21 (4 mg/kg) or saline, administrated by intraperitoneal injection, every 8 h starting at 32 h during hyperoxia. Lungs of these mice were used to prepare nuclear and cytoplasmic extracts. A representative image of Western blot immunoreactive bands of NF-κB p65 in the nuclear extract and IκB in the cytoplasmic extract of lungs of these mice. Bar graph showing the integrated density value of NF-κB p65 in the nuclear extract and IκB in the cytoplasmic extract of mice that received saline (black bar) and GTS-21 (4 mg/kg) (grey bar; n = 5 for saline, and n = 6 for GTS-21 treated mice). β actin expression was measured as a loading control