Figure 6

PsA961 (NOX4^Y512C) derived osteoclasts show enhanced differentiation and increased hydrogen peroxide generation activity compared to a PsA patient (non-carrier of NOX4 rare variants) and to a healthy control.

(A) Osteoclasts were induced through differentiation using colony-stimulating factor (M-CSF/M) and receptor activator of nuclear factor κB ligand (RANKL/R). Differentiated osteoclast with more than three nuclei were identified through tartrate-resistant acid phosphatase (TRAP) staining (violet-labeled). Notably, cells derived from PsA961 patient carrying the NOX4^Y512C variant shown a higher numbers of differentiated osteoclasts (indicated by arrows) compared to age-gender-matched PsA77 patient without NOX4 variants. N = 3. (B) Hydrogen peroxide (H₂O₂) levels, probed by a green dye in cells from PsA961 patients, were significantly higher compared to control cells (C17 and PsA77) at different time points after differentiation with M-CSF and RANKL. Representative images are shown. (C) The average fluorescence intensity was quantified by ImageJ software at three time points. N = 3. (D) In differentiated osteoclast (Day 8) NOX4 protein levels were higher in PsA961 compared to a healthy control—C11. GADPH levels were used as a loading control. N = 3. Data information: H₂O₂ hydrogen peroxide. Scale bars = 100 μm. N =  biological replicates. For graphs (A, C, D), error bars in figures represent mean ± SEM. One-way ANOVA with Tukey’s correction for multiple testing was used. Source data are available online for this figure.