Figure 5.

1-Deoxysphingolipids trigger PYCARD speck assembly. Clonal macrophage reporter (CMR) cells overexpressing fluorescent PYCARD were pretreated with the CASP1 inhibitor before incubation with 1 μM alkyne SA or 1 μM doxSA mix or 10 µM nigericin for 16 h or 90 min (nigericin). If applying cells were also pretreated with cytochalasin D (CyD) or bafilomycin A (Baf) or co-treated with 25 μg/mL FB1. After fixation and staining of nuclei epifluorescence microscopy was performed. (A) Micrographs depict activated PYCARD specks (green) and nuclei (magenta, DRAQ5). Note that activation usually leads to formation of one PYCARD speck per cell. Scale bar, 50 μm. (B) Semi-quantitative analysis of PYCARD speck assembly (% of all cells) upon the various treatments. Mean values ± SEM are plotted. Statistical differences were calculated by ANOVA followed by Dunnett’s multiple comparison test. Adjusted P values: **** p < 0.0001; * p = 0.0121; ns – not significant; all vs. untreated control