Figure 4.

A, BAA473 induces cytokine production via ASC/CASP1 but not NLRP3. LPS-primed (0.1 ng/ml) PBMCs were pretreated with 5 μm BAA473 or 5 mm ATP for 1 h followed by overnight treatment with increasing concentrations of the NLRP3 inhibitor MCC950. IL-1β secretion was measured by HTRF. B–D, genome-wide CRISPR screen to determine the mechanism of action of BAA473. B, screening workflow. C, LPS-primed (1 μg/ml) THP-1 cells were treated with BAA473 overnight, after which cell death was assessed by CTG, whereas IL-1β secretion was measured by HTRF. Induced cell death observed in LPS-primed (1 μg/ml) THP-1 cells upon treatment with BAA473 was used as a readout in the genome-wide CRISPR screen. D, THP-1-Cas9 cells were transduced with a genome-wide lentiviral CRISPR library. Cells were primed with LPS and after 24 h were treated with 75 μm BAA473. The next day, live cells were FACS-sorted using fluorescent live stain. The screen was run in duplicate. Gene-centric visualization is shown as the average log₂ -fold change versus significance score in live cells. Error bars, S.E.