Fig. 6

Fn stimulates NETs generation by activating TLR4-ROS signaling and NOD1/2 receptor. A Flow cytometry analysis for ROS levels in neutrophils treated with Fn or PMA for 8 h. The mean fluorescence intensity (MFI) of three independent experiments is quantified and shown below. B Representative IF images for DNA / NE / MPO in neutrophils treated with Fn or Fn + DPI for 8 h. C Western blot analysis of CitH3 and PAD4 in neutrophils treated with Fn or Fn + DPI for 8 h. Protein levels were quantified by using densitometry and normalized to β-actin and are shown as fold changes compared to the control (Right). Each bar displays the means ± SD of three independent experiments. D ELISA analysis was performed to detect MPO-DNA levels in the supernatant of neutrophils treated with Fn or Fn + DPI for 8 h. E Representative IF images for DNA / NE / MPO in neutrophils treated with Fn or Fn + TAK-242 for 8 h. F Western blot analysis of CitH3 and PAD4 in neutrophils treated with Fn or Fn + TAK-242 for 8 h. Protein levels were quantified by using densitometry and normalized to β-actin and are shown as fold changes compared to the control (Right). Each bar displays the means ± SD of three independent experiments. G ELISA analysis was performed to detect MPO-DNA levels in the supernatant of neutrophils treated with Fn or Fn + TAK-242 for 8 h. H Flow cytometry analysis was used to detect ROS levels in neutrophils treated with Fn or Fn + TAK-242 for 8 h. The MFI of three independent experiments is quantified and shown (Right). I Representative IF images for DNA / NE / MPO in neutrophils treated with Fn, Fn + ML130 or Fn + GSK717 for 8 h. J Western blot analysis of CitH3 and PAD4 in neutrophils treated with Fn, Fn + ML130 or Fn + GSK717 for 8 h. Protein levels were quantified by using densitometry and normalized to β-actin and are shown as fold changes compared to the control (Right). Each bar displays the average + SD of three independent experiments. K ELISA analysis was performed to detect MPO-DNA levels in the supernatant of neutrophils treated with Fn, Fn + ML130 or Fn + GSK717 for 8 h. L Flow cytometry analysis was used to detect ROS levels in neutrophils treated with Fn, Fn + ML130 or Fn + GSK717 for 8 h. The MFI of three independent experiments is quantified and shown (Right). None, no treatment. White scale bars: 50 μm. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001