Figure 8.

LC3 is required for activation of MAPKs-AP-1 pathway and cytokine production upon H. capsulatum stimulation. (A–D) Macrophages from WT mice were transfected with control siRNA or siRNA against LC3α/β (25 nM) for 72 h before stimulation with H. capsulatum [MOI = 2 (A) and 5 (B,C)]. For (A), macrophages were washed to rid of unengulfed yeasts at 1 h after stimulation then lysed immediately (0 h) or after 18 h of incubation, and the number of yeast cells were counted. Replication time (h) = incubation interval/number of divisions (n = 5). For (B), culture supernatants were collected at 18 h after stimulation, and the concentrations of TNF, IL-6, and IL-β in the supernatants were quantified by ELISA and are presented as the relative levels of each cytokine (n = 6 for TNF and IL-6; n = 4 for IL-1β). For (C), cell lysates were collected at 1 h after stimulation and analyzed by Western blotting for the expression of indicated proteins. Relative intensity of indicated protein normalized against the corresponding β-actin was shown in (D) (n = 3). (E,F) Macrophages from WT mice were treated with or without DPI (5 μM) for 1 h prior to stimulation with H. capsulatum (MOI = 5). Cell lysates were collected at 1 h after stimulation and analyzed by Western blotting (E). Culture supernatants were collected at 18 h after stimulation, and the concentrations of TNF, IL-6, and IL-β in the supernatants were quantified by ELISA (n = 4) (F). Bars represent the mean ± SEM, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 [2-tailed t-test (A,B, D); ANOVA with Bonferroni's multiple comparisons post-hoc test (F)].