Fig. 4: Murine Adenovirus 3 dampens various inflammasome responses in ASC-dependent manner.
A LDH assay in RAW264.7 macrophages, iBMDMs (IFNγ-primed) and iBMDMs (unprimed) after viral pre-infection (for 8 h) with increasing MOIs of mAdV3, as indicated and bacterial secondary infection with STm (right-hand three bars for each cell line) at an MOI of 50 for 16 h. Colors indicate whether co-infection showed a synergistic (blue), antagonistic (orange) or neutral (gray) trend. Each point represents a technical replicate across three biological replicates, bars depict the mean and error bars indicate standard deviation. For statistical assessment, two-sided Student’s T-test with Welsh correction was used. B GO-term enrichment of proteins with an absolute log2-fold-change larger than 0.5, p value < 0.05. GO-term overrepresentation was performed without correction and only terms with an enrichment >3, at least 3 proteins, and p value < 0.01 (Fisher’s exact test, for enrichment only) were selected. Bubble size indicates the number of mAdV3-up- or down-regulated proteins for each term. C Cell death as quantified by LDH release for BMDMs that were pre-infected with mAdV3 for 8 h (dark bars) or not (light bars), and subsequently treated with the indicated inflammasome activator for 2 h. p values were obtained by two-sided Student’s T-test with Welsh correction and bars represent mean with SEM, n = 254. D As in panel C, but with IL−1ß released, measured by ELISA, n = 218. Orange colored bars indicate antagonisms, gray bars represent neutral interactions. E Representative fluorescence microscopy images (crop from a field of view acquired with 20x magnification) after staining with ASC-antibody (red) and DAPI (blue) of cells treated as described in panel C. Arrows indicate ASC-speck positive cells, scale bar: 20 μm. F Quantification of 120 fields of view, represented by the example given in panel E. Coloring and statistics as in panel C, bars represent mean with SEM. For consistency purposes p = 0.0002 is displayed as p < 0.001.
