Fig. 5: Apaf-1 recruits RIP2 to mediate inflammatory responses upon cytoplasmic DNA sensing. | Cell Discovery

Fig. 5: Apaf-1 recruits RIP2 to mediate inflammatory responses upon cytoplasmic DNA sensing.

From: Apaf-1 is an evolutionarily conserved DNA sensor that switches the cell fate between apoptosis and inflammation

Fig. 5

a HEK293T cells were co-transfected with HA-tagged Apaf-1 and plasmids expressing Flag-tagged proteins containing death domains (DD). Proteins were immunoprecipitated with anti-HA antibody, and bound proteins were detected with anti-Flag or anti-HA antibody. b–d Protein complex of Apaf-1 and RIP2 in HEK293T and cGas−/− HeLa cells, which were transfected with HT-DNA (3 μg/mL) for the indicated times. Immunoblot analysis of endogenous Apaf-1 and RIP2 immunoprecipitated with antibodies against Apaf-1 or RIP2. e WT and Apaf-1−/− A549 cells were transfected with HT-DNA as indicated. Cell lysates were separated by native PAGE or SDS‒PAGE and analyzed by immunoblotting with the indicated antibodies. f The cGas−/− HeLa cells expressing both mCherry-Apaf-1 and EGFP-RIP2 were transfected with HT-DNA or HSV60. At 4 h post-transfection, cells were fixed, stained with DAPI, and imaged by confocal microscopy. Scale bars, 10 μm. g HA-tagged full-length or truncated Apaf-1 expression plasmids were coexpressed with Flag-tagged RIP2 in HEK293T cells for 24 h and then infected with HSV-1 for another 4 h. After incubation with anti-HA beads, the bound proteins were analyzed by immunoblotting with anti-Flag or anti-HA antibodies. h Flag-tagged RIP2 or truncated expression plasmids were coexpressed with HA-tagged Apaf-1 in HEK293T cells for 24 h and then transfected with HT-DNA for another 4 h. After incubation with anti-HA beads, the bound proteins were analyzed by immunoblotting with anti-HA or anti-Flag antibodies. i Expression plasmids (50, 200, and 1000 ng) of Flag-tagged human RIP2 were transfected into WT or Apaf-1−/− A549 cells for 24 h and then treated with HT-DNA (3 μg/mL) or HSV-1 (MOI = 3) for 12 h. The expression of TNF-α or RIP2 was measured by qRT‒PCR. j Heatmap of the induction of TNF-α, IL-1α, IL-1β, CCL4, and PTGS2 mRNA in WT and two different Rip2−/− A549 cells transfected with HT-DNA (3 μg/mL) for the indicated times. k Heatmap of indicated pro-inflammatory cytokine and chemokine expression profiles in full-length or truncated RIP2 reconstitution in Rip2−/− A549 cells with HT-DNA stimulation for 12 h. Mean ± SEM of triplicates, P values were calculated using a two-tailed unpaired Student’s t-test.

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