ERMES impacts on the exposure of pathogen-associated molecular patterns during hyphal growth. ERMES impacts on the exposure of pathogen-associated molecular.

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ERMES impacts on the exposure of pathogen-associated molecular patterns during hyphal growth. ERMES impacts on the exposure of pathogen-associated molecular patterns during hyphal growth. (A) Hyphae were grown for 3 h in RPMI medium under repressive conditions, and surface-exposed 1,3-β-glucan was analyzed by flow cytometry following staining with the 1,3-β-glucan antibody. The experiment was repeated 3 times with equivalent results (see also panel B). Shown are representative dot plots from one biological replicate for each strain, plotting 1,3-β-glucan staining versus cell size (forward scatter [FSC]), with the percentage of cells shown for each quadrant. The upper left and right quadrants are glucan-positive cells, while the lower left and right quadrants are glucan-negative cells. (B) Percentage of cells that are negative for exposed 1,3-β-glucan. n is 9 from 3 independent experiments with 3 biological replicates each. The biological replicates analyzed together are shown by the same color. The line represents the mean. *, P < 0.05; **, P < 0.01 (one-way analysis of variance, followed by Tukey’s multiple-comparison test). (C) Hyphal length after 3 h in RPMI medium. Each measured cell is depicted by a line, with length shown on the y axis and the measurements ranked in order from smallest to largest on the x axis. Three biological replicates were performed. One repeat is shown here, and the other two are in Fig. S7B in the supplemental material. (D) Hyphal gene expression after 3 h in repressive RPMI medium. Shown is the ratio of gene expression normalized to SCR1. Error bars represent standard errors of the averages from 6 biological replicates assayed in 2 independent experiments. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 (one-way analysis of variance followed by Tukey’s multiple-comparison test). (E) Hyphal length distribution in macrophages following 1 h of coincubation and washes. Data were analyzed in 2 independent experiments with equivalent results (n = 200 fungal cells per strain). One experiment is shown here, and the other is shown in Fig. S7C in the supplemental material. (F) Growth at 37°C in repressive medium. Shown are the averages and the standard errors of the means from 3 biological replicates assayed in the same experiment. OD600, optical density at 600 nm. Timothy M. Tucey et al. mSphere 2016; doi:10.1128/mSphere.00074-16