Figure 2.

NLRP1 regulated pyroptosis in primary cortical neurons in the presence of Aβ. Knockdown of NLRP1 in primary cortical neurons. Primary cortical neurons were transfected with NLRP1 siRNA or control siRNA for 48 h. After transfection, the cells were then incubated in the presence or absence of 5 μM Aβ for 24 h. (a) The expression levels of NLRP1 were determined by western blot analysis and quantified by densitometric measurement. β-Actin was used as a loading control. (b) Cell viability was determined by MTT assay and shown as a percentage of surviving cells. (c) DNA breaks were analyzed by TUNEL assay; and cell nuclei were stained with DAPI. Representative images of DAPI-stained and TUNEL-positive cells were shown. Scale bars: 20 μm. (d) Quantification for the percentage of TUNEL-positive cells. (e) The release of caspase-1 (procaspase-1 p45 45 kD and cleaved caspase-1 p20 subunit 20 kD) and cleaved IL-1β to the culture medium was examined by immunoblotting. β-Actin was used as a loading control. (f) Levels of cleaved caspase-1 p20 were quantified by densitometric measurement. (g) The presence of IL-1β in cell culture supernatants was measured by ELISA. (h) The release of LDH into culture supernatants and expressed as the percentage of LDH release compared with a maximum lysis control. (i) Typical result of Aβ untreated and treated cells. Fluorescent microscopy images showing the bright, small speck-like, perinuclear localization of ASC foci (green) with nuclei visualized by DAPI staining (blue) in the right panel. Scale bars: 10 μm. All values are presented as the mean±S.E.M. of three independent experiments. *P<0.05 versus control siRNA treatment