FIGURE 7.

ABC-induced inflammasome activation is initiated upstream of mROS production and requires cardiolipin. A and B, flow cytometric analysis of the percentage of MitoSOX positivity (A) or MitoSOX median fluorescence intensity (MFI MitoSOX) (B), showing mROS production of TPA-primed wild-type THP-1 cells (THP1-WT) and NLRP3-deficient THP1 cells (THP1-defNLRP3) upon treatment with medium (ctrl), or 0.5 mg/ml ABC. C, left, analysis of medium- (ctrl), ABC-, or ATP-induced IL-1β release from TPA-primed THP1 after transfection with a scrambled siRNA (si scrambled) or a pool of two siRNAs against cardiolipin synthase 1 (si CRLS1). Right, evaluation of siRNA-mediated CRLS1 mRNA knockdown and its impact on TPA-mediated IL-1β mRNA induction as quantified by qRT-PCR. Bar diagrams show mean values ± S.D. of n ≥ 3 experiments with qRT-PCR data in C presented as -fold GAPDH-normalized expression of the indicated mRNAs in relation to the respective TPA-treated si scrambled control arbitrarily set to 1. Histograms in A are representative of n = 3 experiments with overlays showing logarithmic MitoSOX (PE-A) fluorescence intensity obtained for live-gated populations of the indicated ABC-stimulated cell line (bold red line), an unstained THP1-WT control (ctrl unstained, gray dotted line), and the respective unstimulated MitoSOX-stained control (ctrl MitoSOX, black thin line), respectively. Indicated percentages denote MitoSOX positivity among the ABC-exposed live population (*, p < 0.05, **, p < 0.01, ***, p < 0.001, one-sample t test (C, right) or two-way ANOVA (B and C, left), Sidak's multiplicity correction).