Fig E2.

T3SS selection and validation of NLRC4 KO THP-1 cells. HEK293T cells were transfected with WT NLRC4, as well as increasing amounts of human NAIP (hNAIP) and the T3SS needle protein PrgI. A, Flow cytometric analysis of ASC speck formation shows that WT NLRC4 is needed for ASC speck formation and that hNAIP is required for ASC specks to increase in response to low amounts of PrgI. B, Various T3SS proteins were transfected into HEK293T cells in addition to WT NLRC4 and hNAIP to ensure specificity of response. The ASC speck response is specific to the only needle protein tested, PrgI. C, THP-1–Cas9 cells infected with inducible single guide RNA targeting exon 2 of NLRC4 were treated with doxycycline for 72 hours and expression of NLRC4 assessed with Western blotting performed on whole-cell lysates after 48 hours of rest. Cells were then primed with Pam3CSK4 and infected with 2 amounts of retrovirus expressing PrgI needle protein. D-F, After 24 hours of assessment of cell death (Fig E2, D), IL-1β (Fig E2, E) and IL-18 (Fig E2, F) secretion was undertaken.