FIG 4 .

Hog1 signaling is required for induction of macrophage pyroptosis via transcriptional regulation of cell wall genes. (a) Levels of ASC speck formation for mutants of genes in the Hog1 cascade. GRACE strains were incubated with 0.5 µg/ml DOX overnight and during infection to repress target gene expression. Macrophages were infected at an MOI of 1:3. ASC specks were quantified in ASC-mCherry macrophages after 4 h of infection from the average results from two technical replicates from at least two biological replicates. All mutants were significantly (P < 0.0001) different from the parent strains, as determined by 1-way ANOVA with Dunnett’s multiple-comparison test. Error bars represent standard deviations. (b) Hog1 is required for transcription of C. albicans genes encoding pyroptosis activators in macrophages. RNA was collected from the indicated strains after incubation with macrophages for 1 h. Significance was determined using 1-way ANOVA. ****, P < 0.0001; ***, P < 0.005; *, P < 0.05. Error bars represent standard errors of the means. (c) Hog1 is required for cell wall remodeling. Cells were incubated with macrophages for 1 h before collection and staining with concanavalin A for mannose. Bar, 10 µm. (d) Quantification of concanavalin A staining after incubation in macrophages for 1 h. Fluorescence levels were quantified using ImageJ from two biological replicates. Significance was determined by unpaired t tests. **, P < 0.01.