To confirm that escape from double-membrane vacuoles during secondary infection was necessary for NLRP6 activation, we compared NLRP6-expressing GFP-ASCtg cells infected with wild-type, Δhly, ΔactA and ΔplcB Listeria, and found that ΔplcB Listeria induced reduced levels of ASC-speck formation in the infected cell population, mimicking the effects of ActA deficiency (Fig. 5H). The gene discussed is ACTA1; the disease is infection.