Fig. 2: Combined deletion of PANoptosis molecules inhibits cell death induced by LPS plus HS. | Nature Communications

Fig. 2: Combined deletion of PANoptosis molecules inhibits cell death induced by LPS plus HS.

From: NINJ1 mediates inflammatory cell death, PANoptosis, and lethality during infection conditions and heat stress

Fig. 2

A–C Immunoblot analysis of A pro- (P45) and activated (P20) caspase-1 (CASP1); pro- (P43) and cleaved (P36 and P26) caspase-11 (CASP11); pro- (P53), activated (P30), and inactivated (P20) gasdermin D (GSDMD); pro- (P53) and activated (P34) gasdermin E (GSDME); B pro- (P55) and cleaved (P44 and P18) caspase-8 (CASP8); pro- (P35) and cleaved (P19 and P17) caspase-3 (CASP3); pro- (P35) and cleaved (P20) caspase-7 (CASP7); C phosphorylated receptor-interacting serine/threonine kinase 3 (pRIPK3), total RIPK3 (tRIPK3), phosphorylated mixed lineage kinase domain-like (pMLKL), and total MLKL (tMLKL) in wild type (WT) bone marrow-derived macrophages (BMDMs) treated with lipopolysaccharide (LPS) alone or subjected to heat stress (HS; 43 °C for 30 min) with or without LPS priming. M, media control. Actin is used as the internal control. D Real-time analysis of cell death in LPS-primed WT, Casp1–/–Casp8–/–Ripk3–/– (TKO), and Casp1–/–Casp11–/–Casp8–/–Ripk3–/– (QKO) BMDMs challenged with HS (43 °C for 30 min). E–G Immunoblot analysis of E pro- (P45) and activated (P20) CASP1; pro- (P53), activated (P30), and inactivated (P20) GSDMD; pro- (P53) and activated (P34) GSDME; F pro- (P55) and cleaved (P44 and P18) CASP8; pro- (P35) and cleaved (P19 and P17) CASP3; pro- (P35) and cleaved (P20) CASP7; and G pRIPK3 and tRIPK3 in LPS-primed WT and QKO BMDMs at the indicated timepoints after HS (43 °C for 30 min). Actin is used as the internal control. H Immunofluorescence images of WT LPS-primed BMDMs at 12 h post-treatment (hpt) (43 °C for 30 min). Nuclei were stained with DAPI. Arrowheads indicate the colocalized ASC, RIPK3, and CASP8 specks. Images are representative of three independent experiments. I Distribution of ASC+ specks colocalized with RIPK3+ or/and CASP8+ specks in (H). n > 100 specks were counted. A–C, E–H Images are representative of at least three independent experiments. D Data are shown as mean ± SEM; **P < 0.01, and ****P < 0.0001 (one-way ANOVA with Bonferroni’s multiple comparisons test; n = 4 from 4 biologically independent samples). I Data are shown as mean ± SEM (n = 9 from 4 biologically independent samples). H Scale bar, 5 μm. Exact P values are presented in Supplementary data file 2. For uncropped western blots, see the accompanying source data.

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