Fig. 4: Genome-wide CRISPR screen identifies NINJ1 as a cell death executioner in response to LPS plus heat stress. | Nature Communications

Fig. 4: Genome-wide CRISPR screen identifies NINJ1 as a cell death executioner in response to LPS plus heat stress.

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

Fig. 4

A Volcano plot showing the genes that are enriched or depleted in immortalized bone marrow-derived macrophages (iBMDMs) after a genome-wide CRISPR-Cas9 knockout screen of cell death induced by lipopolysaccharide (LPS) priming and heat stress (HS; 43 °C for 60 min). B Analysis of the enrichment of genes encoding pore-forming molecules identified in (A) in the CRISPR screen results. C NINJ1 expression in MCF7, MDA231, and MDA468 cancer cells with and without hyperthermia (GSE48398). D Immunoblot analysis of NINJ1 expression in wild type (WT) BMDMs after 2 or 12 h of LPS stimulation or following HS challenge (43 °C for 60 min). E Real-time analysis of cell death under basal conditions (no LPS + HS) or in LPS-primed WT, Ninj1–/–, and glycine (Gly)-treated WT BMDMs following challenge with HS (43 °C for 30 min). F Representative images of cell death in (E) at 16 h post-treatment (hpt). G Real-time analysis of cell death under basal conditions (no HS) or in WT, Ninj1–/–, and Gly-treated WT BMDMs following challenge with HS (43 °C for 60 min). H Representative images of cell death in (G) at 16 hpt. I Analysis of cell death in human macrophages 5 h after LPS + HS (43 °C for 60 min) treatment or HS (43 °C for 2 h) treatment in the presence or absence of Gly. J Real-time analysis of cell death in LPS-primed WT and Gsdmd–/–Gsdme–/–Mlkl–/– BMDMs and Gly-treated WT, Gsdmd–/–, Gsdme–/–, Mlkl–/–, Gsdmd–/–Gsdme–/–, Gsdmd–/–Mlkl–/–, and Gsdmd–/–Gsdme–/–Mlkl–/– BMDMs following challenge with HS (43 °C for 30 min). B Data are shown as mean. The detailed data analysis used for panels A and B is described in the “Methods”. C Data are shown as mean ± SEM; *P < 0.05, **P < 0.01 (two-tailed t-test; n = 3–6 samples from GSE48398). D, F, H Images are representative of at least three independent experiments. F, H Scale bar, 50 μm. E, G, J Data are shown as mean ± SEM; **** 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; ***P < 0.001 (two-tailed t-test; n = 3 from 3 biologically independent samples). Exact P values are presented in Supplementary data file 2. For uncropped western blots, see the accompanying source data.

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