Figure 6

CATH-1 negatively regulates NF-κB and ERK signaling pathways in SS2-infected macrophages. Macrophages were incubated with CATH-1 for 2 h (pre-incubation). Then, cells were washed with RPMI twice and then infected with SC19 for 2 and 6 h. At 2 and 6 hpi, gentamicin (250 μg/mL) was added and incubation continued for 22 and 18 h. After 24 h incubation, the supernatants were collected to determine the production levels of IL-1β, IL-6, and TNF-α (A and C). Meanwhile, at 2 hpi, cells of partial wells were directly lysed by 0.1% Triton X-100 to quantify adherent bacteria and gentamicin was added to another aliquot of cells for 30 min before lysis to quantify intracellular bacteria (B). Macrophages were incubated with or without CATH-1 for 2 h (pre-incubation). Subsequently, cells were washed with Opti-MEM twice and infected with SC19 at an MOI of 5 for 0, 15, 30, 60, and 120 min to determine expression of phosphorylation of NF-κB p65 and ERK1/2 (D). Data are represented as mean ± SEM of three independent experiments of triplicate samples per experiment. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, ns, no significance.