Figure 3

Extracellular NLRP3-YFP inflammasome particles promote release of pro-atherogenic cytokines and cell migration in HCASMC. (A, B) Supernatant of HCASMC (N = 3) after 4 h treatment with extracellular NLRP3-YFP inflammasomes (3:1 particles/cell) were used for the human proteome profiler cytokine array. Modified cytokines/chemokines were surrounded with a red square and labelled (1–7). Membranes were applied for short and long-time exposure. (B) Densitometric analysis of the labelled cytokines (1–7) after background subtraction was performed. Data were normalized on supernatant from control cells which was set at 1. (C) Scratch assay of HCASMC treated with NLRP3-YFP inflammasome particles (3:1 particles/ cell) for 4 h. PDGF (10 ng/ml) and Actinomycin (5 µg/ml) were stimulated for 24 h. Representative images of wounds at the beginning (0 h) and after 24 h are shown. Wounds were automatically acquired within the CO₂ incubator using the IncuCyte ZOOM software package. (D) Cell count [N] at 0 h and after 24 h within the scratch and (E) wound confluency [%] was calculated using the IncuCyte ZOOM software. Data are expressed as mean ± SEM and differences between the groups were calculated using unpaired, two-tailed Student´s T-Test (*p < 0.05). (F) Smooth muscle cell migration was studied in a Boyden chamber by adding NLRP3-YFP inflammasome particles (3:1 particles/ cell) to HCASMC for 4 h and analyzing the migrated cells on the lower side of the insert membrane which was stained with crystal violet and measured at 560 nm in a fluorometer (N = 4–6). Differences between the groups were analysed by One-way ANOVA and uncorrected Fishers LSD post-hoc test (*p < 0.05).