FIGURE 2.

Silica remodels ETC complexes activity. RAW 264.7 macrophages were exposed to LPS (1 ng/ml) or silica (50 μg/cm²) or primed with LPS (1 ng/ml for 24 h) and then exposed to silica (50 μg/cm²) at time points of T0, 2, 4, 6, and 24 h. RAW 264.7 macrophages (5 × 10⁶/chamber) were allowed to equilibrate in the chamber for ∼10 min before measuring oxygen flow. Default respirometric settings of block temperature 37°C, stir bar speed 400 rpm, and data recording every 2 s were used. Oxygen turnover (oxygen concentration, left y-axis; oxygen flux, right y-axis) by treated RAW 264.7 macrophages was followed after addition of (A) succinate (final concentration [f.c.], 10 mM) or (C) rotenone (f.c., 0.5 µM), succinate (f.c., 10 mM), ADP (f.c., 2.5 mM), CCCP (f.c., 0.05 µM), and antimycin A (f.c., 2.5 µM). After the addition of each compound, cells were allowed to come to equilibrium. Because the variation in oxygen concentration (broken traces) is difficult to appreciate because it is very little, the slope of the oxygen concentration is also shown (solid trace), which represents the negative derivative of the oxygen concentration. Variations in oxygen flux for each condition were measured as a ratio between the value of the O₂ slope at the equilibrium after treatment with succinate (with or without prior inhibition of complex I with rotenone), and the value of O₂ slope at the baseline. (B) Fold increase of oxygen flux after stimulation with succinate. (D) Fold increase of oxygen flux after the inhibition of CI with rotenone and stimulation of CII with succinate. (E) The production of H₂O₂ has been assessed following the slope (purple trace) of the concentration of the fluorescent Amplex Red assay product resorufin (green trace) while performing a SUIT assay protocol (see Substrate-uncoupler-inhibitor titration assay.). Variations in H₂O₂ for each condition were measured as the ratio between the value of the slope at the equilibrium after the inhibition of CI with rotenone and the stimulation of CII with succinate, the slope at the equilibrium at the baseline. (F) Fold increase of H₂O₂ production after inhibition of CI with rotenone and stimulation of CII with succinate. n = 4; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus control. Sil, silica.