Figure 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 µmol/L) or remained at 21% O2 for 24 h. After oxygen exposure, cells were washed with PBS, fixed, permeabilized and stained to localize the NF-κB p65 subunit (red). Multiple pictures were taken using an immunofluorescence microscope to visualize the localization of the p65 subunit of NF-κB. Counterstaining with DAPI was used to visualize nuclei (blue). The immunofluorescence images shown are representative of three independent experiments. (B) 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, administered by intraperitoneal injection every 8 h starting at 32 h during hyperoxia. Lungs of these mice were used to prepare nuclear and cytoplasmic extracts. Representative images are shown 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 shows 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 in GTS-21-treated (4 mg/kg) mice (gray bar) (n = 5 for saline, and n = 6 for GTS-21-treated mice). β-Actin expression was measured as a loading control.