Journal: Nature Communications
Article Title: RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL
Figure Lengend Snippet: XIAP is required to repress LPS- and TNF-induced IL-1β secretion. ( a – f ) WT and Xiap -deficient ( x −/− ) macrophages were pre-incubated with or without ( a – c ) LPS (20 ng ml −1 ) or ( d – f ) human Fc-TNF (100 ng ml −1 ) for 2–3 h and cultured with or without IAP antagonists of differing IAP specificities (see g ). After 24 h, cell supernatants were assayed for ( a , d ) IL-1β, ( b , e ) TNF and ( c , f ) IL-6 levels by ELISA. n =3 mice; Data are represented as mean+s.e.m., from one of three experiments. ( g ) Efficiency of functional XIAP antagonism by IAP antagonist compounds (+, high; −,low). ( h ) WT, cIAP1 −/− ( c1 −/− ), cIAP2 −/− ( c2 −/− ) and Xiap −/− ( x −/− ) BMDM were primed with LPS (20 ng ml −1 ) for 3 h and cultured with the IAP antagonist LBW242 (20 μM) or alum (320 μg ml −1 ) for a further 6 h. Secreted IL-1β was measured in supernatants by ELISA. n =3 mice; mean+s.e.m. ( i ) Yield of macrophages from WT and IAP mutant bone marrow after 6 days of culture with L929 cell conditioned media. n =3–6 mice per genotype, mean+s.e.m. ( j – l ) WT and IAP mutant macrophages were stimulated with LPS (20 ng ml −1 ) for up to 24 h, and ( j ) IL-1β, ( k ) TNF and ( l ) IL-6 levels were assayed in supernatants by ELISA. n =3–4 mice, data are represented as mean+s.e.m., one of three experiments. ( m ) Yield of WT, c1 lox/lox x −/− c2 −/− , c1 LysMcre x −/− c2 −/− and c1 ERcre x −/− c2 −/− bone marrow macrophages after 6 days of culture with L929 cell conditioned media. n =3–6 mice per genotype, mean+s.e.m. ( n , o ) WT, c1 lox/lox x −/− c2 −/− , c1 LysMcre x −/− c2 −/− and c1 ERcre x −/− c2 −/− macrophages were pulsed for 16 h with 4′-hydroxy-tamoxifen (4HT 1000, nM) and then rested for 10 h prior to stimulation with or without LPS (50 ng ml −1 ) for a further 8 h. ( n ) Secreted IL-1β was measured in supernatants by ELISA, n =3 mice per group; c1 LysMcre x −/− c2 −/− ( n =2), mean+s.d., one of three experiments, and ( o ) IL-1β and caspase-1 activation assayed by immunoblot of supernatants and lysates. Representative blot from the analysis of 4 c1 ERcre x −/− c2 −/− mice. Full-size immunoblots are presented in Supplementary Fig. 9 .
Article Snippet: Cytokine ELISA IL-1β and IL-18 (R & D Systems, Ebioscience), and TNF and IL-6 (Ebioscience, R & D Systems) ELISA kits and paired G-CSF antibodies and standard (R & D Systems, Peprotech) were used to perform ELISAs on serum, joint fluid and supernatants according to the manufacturer’s instructions.
Techniques: Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay, Mouse Assay, Functional Assay, Mutagenesis, Activation Assay, Western Blot