Fig. 3.

Nox4-dependent H₂O₂ production in subclonal expanded p22phox-CRISPR/Cas9 knockout Nox4/tetNox4-HEK293 cells. Either Nox4-HEK293 (A) or induced tetNox4-HEK293 cells (B) were transfected with empty vector (Vec) or gRNA combination 1 or 10, selected with puromycin and subclonally expanded. Each clone is named according to the vector control (Vec) or gRNA combination (1 or 10) and ongoing number (e.g. C1-1). Relative luminol/HRP assay was performed in subclonally expanded control cells (Vec) or CRISPR/Cas9-mediated p22phox knockout (combinations 1 or 10) Nox4-HEK293 (A) or tetNox4-HEK293 cell lines (B). Also shown are representative Western blots for p22phox, Nox4 and β-Actin. n≥3, mean±SEM, ***p<0.001 relative to corresponding vector clones.