Figure 4.

Ca²⁺-NFAT-IL-2 axis is affected by early autophagic events, phagocytosis and LAP. nuclear factor of activated T cells translocation and IL-2 production of NFAT-luciferase reporter D1 cells that have been stimulated with A-sw for 6 h in the presence and absence of drugs inhibiting specific cellular processes. (A) Fungus-stimulated D1 cells in the presence and absence of the type III Phosphatidylinositol 3-kinase inhibitor 3MA, 10 mM, or the vacuolar H + ATPase inhibitor, Baf (50 nM), which inhibits the early and late stage of autophagy respectively. (B) Fungus-stimulated D1 cells in the presence or absence of the phagocytosis inhibitor, CytoD (2 µg/mL). (C) D1 cells stimulated with fungi in the presence or absence of the lysosomal acidification inhibiting combination of Leu/A. Data is displayed as the mean ± SD of five (in the case of Leu/A) or eight biological replicates. (D) Viability of D1 cells treated with fungus, drugs, or a combination of the both. (E) mRNA expression of IL-2 in D1 cells silenced for various mRNA regulating LAP in the presence or absence of A. fumigatus swollen conidia. Data is displayed as means ± SD of three biological replicates. Differences found to be statistically significant by one-way ANOVA with Bonferroni’s Multiple Comparison post-test are indicated (NS, non-significant; ***, p < 0.001; ****, p < 0.0001). Abbreviations: Untreated (Unt); A. fumigatus swollen conidia (A-sw); Vehicle non-treated control (NT); 3-methyladenine (3MA); Bafilomycin (Baf); Cytochalasin D (CytoD), combination of Leupeptin and ammonium chloride (Leu/A).