FIGURE 3.

Silica inhibits CI activity in part by reducing Ecsit expression. 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) and examined as a function of time 0–24 h. (A and B) Mitochondria pellets were isolated from RAW 264.7 macrophages, and complex I and complex II enzymatic activity were analyzed spectrophotometrically and measured as mAbs/min/μg proteins (see CI assay and CII assay.). n = 4. (C and D) Mitochondria were isolated from RAW 264.7 macrophages exposed to LPS (C) or silica (D) for the indicated times, and Ecsit (37-kDa subunit) abundance was determined by Western blot while using complex IV subunit MTCO1 as markers for mitochondria and GAPDH expression as a loading control. (E) Confocal microscopy of RAW 264.7 macrophages stimulated for 4 h with LPS (1 ng/ml) or silica (50 μg/cm²) in the presence or absence of LPS priming. Mitochondria are visualized by staining with ATPase (red), DNA into the nuclei of cells with Hoechst (blue), followed by the analysis of Ecsit (green). Scale bar: 10 μm. (F) Graph illustrating the abundance of ESCIT in LPS- or silica-exposed RAW 264.7 macrophages. Data are expressed as the mean ± SEM and are representative of three different experiments. *p < 0.05, ***p < 0.001, LPS versus silica; ^##p < 0.01, ^###p < 0.001 versus baseline. CTR, control.