Nox4 mediates IL-1ß-effects on MMP-1 secretion and DNA fragmentation in C-20/A4 chondrocyte cells.

<p>(<b>A</b>) Predicted linear domain structure of Nox4A, the functional isoform and Nox4B, deleted for a putative NADPH binding site. (<b>B</b>) ROS production was measured by chemiluminescence on 5×10⁵ intact C-20/A4 chondrocytes overexpressing Nox4A or Nox4B treated or not with 5 mM of Tiron. Results are expressed as the sum of all RLU measurements recorded every 30 s during 45 min. Values represent the mean +/− S.D. of four determinations obtained the same day. * p<0.05 versus untreated Nox4A cells. (<b>C</b>) C-20/A4 chondrocytes transfected with genes encoding for Nox4A or Nox4B were stimulated or not with 2 ng/ml IL-1ß with or without 5 mM Tiron. After 48h, supernatant was collected, concentrated 10 times by centricon and 10 µg of proteins were loaded for MMP-1 immunodetection by Western Blot. Results are representative of three independent experiments. (<b>D</b>) C-20/A4 chondrocytes transfected with genes encoding for Nox4A or Nox4B were treated or not with 10 ng/ml IL-1ß. After 5 days, cells were detached and fixed with ice cold absolute ethanol. Cells were then washed twice in PBS and stained with propidium iodide before flow cytometry acquisition. * p<0.05 versus Nox4B cells.