FIGURE 8.

Malonylation of GAPDH correlates with TNF-α production in LPS-treated macrophages, while decreased itaconate levels correlate with decreased IFN-β in silica-exposed macrophages. (A–C) RAW 264.7 macrophages were stimulated with LPS (1 ng/ml) or silica (50 μg/cm²) with or without priming and examined at time points up to 24 h. (A) Cell supernatants were examined for TNF-α secretion using ELISA. (B) TNF-α and GAPDH mRNA expression levels quantified by RT-PCR. (C) Immunoprecipitated GAPDH from RAW 264.7 macrophages treated as indicated. Samples probed with an anti-malonyl lysine (anti-MALK) Ab (lower panel). GAPDH expression in the immunoprecipitated (upper panel) samples was also examined. (D and E) RAW 264.7 macrophages were treated with LPS (10 ng/ml) or silica (50 μg/cm²) with or without priming. (D) IFN-β mRNA expression levels quantified by RT-PCR in RAW 264.7 macrophages treated as described for the indicated time. (E) ELISA of IFN-β released in supernatant shows that cells exposed to silica or LPS secrete the same amount of cytokine, but silica inhibits the release in primed cells. (F) The intracellular relative amount of itaconate at a steady state is determined by LC-HRMS. Atomic percent enrichment was calculated using the MIMOSA method. Data are the mean ± SEM of six samples, n = 6. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.