FIG 3.

NLRP1B inflammasome activation in response to Listeria infection is dependent on listeriolysin O (LLO). (A) HT1080 cells expressing pro-caspase-1-T7, pro-IL-1β-HA, and NLRP1B were infected with either wild-type (WT) Listeria or Listeria Δhly mutant at an MOI of 50 or were incubated with glucose-free, serum-free DMEM supplemented with 10 mM 2DG for 3 h. The cell lysates were then assayed for ATP. (B) Supernatants of cells as described for panel A were assayed for LDH activity. (C) Supernatants of cells as described for panel A were immunoprecipitated with anti-HA antibodies and then probed for HA-tagged IL-1β by immunoblotting. (D) HT1080 cells expressing pro-caspase-1-T7, pro-IL-1β-HA, and NLRP1B were infected with either wild-type (WT) Listeria or Listeria ΔactA mutant at an MOI of 50 or were incubated with glucose-free, serum-free DMEM supplemented with 10 mM 2DG for 3 h. The cell lysates were then assayed for ATP. (E) Supernatants of cells as described for panel D were assayed for LDH activity. (F) Supernatants of cells as described for panel D were immunoprecipitated with anti-HA antibodies and then probed for HA-tagged IL-1β by immunoblotting. Blots are representative of three independent experiments. Cross-reacting bands were detected between 25 and 40 kDa. Graphed data represent means ± standard deviations from three independent experiments.