Fig 5.

Effects of p.W655C NLRC4 on cytokine production and cell death. NLRC4 KO THP-1 monocytes were transduced with lentivirus coding for WT or various NLRC4 mutations (as indicated). Twenty-four hours after transduction, cells were plated and treated with Pam3CSK4. A-C, After 24 hours, propidium iodine (PI) staining was assessed by using flow cytometry to quantify cell death (Fig 5, A), and IL-1β (Fig 5, B) and IL-18 (Fig 5, C) secretion was measured by means of ELISA. D, Expression of NLRC4 under each condition was qualified by means of Western blotting of whole-cell lysates. E-H,CASPASE1, PYCARD, or CASPASE8 KO THP-1 monocytes were transduced with WT or p.W655C NLRC4, and assessment of cell death (Fig 5, E) and IL-1β (Fig 5, F) and IL-18 (Fig 5, G) secretion was undertaken after treatment with Pam3CSK4, with expression of NLRC4 determined by using Western blotting (Fig 5, H). THP-1 cells, along with NLRC4, CASPASE1, PYCARD, or CASPASE8 KO THP-1 monocytes, were primed for 3 hours with Pam3CSK4 and infected with 2 amounts of retrovirus expressing PrgI needle protein. I-K, After 24 hours, cell death (Fig 5, I) and IL-1β (Fig 5, J) and IL-18 (Fig 5, K) secretion were assessed with flow cytometry and ELISA, respectively. Data were pooled from at least 3 independent experiments. *P < .05, **P < .01, and ***P < .001.