Skip to main content
. 2022 Dec 5;11:e78609. doi: 10.7554/eLife.78609

Figure 1. NINJ1 knockout functionally and morphologically phenocopies glycine cytoprotection.

(A) Immunoblot analysis demonstrating NINJ1 knockout in immortalized bone marrow-derived macrophage (iBMDM). GAPDH(glyceraldehyde-3-phosphate dehydrogenase) is presented as a loading control. (B–D) Wildtype and NINJ1 knockout iBMDM were induced to undergo pyroptosis (LPS + nigericin), post-apoptosis lysis (secondary necrosis; venetoclax, ABT-199), or necrosis (pneumolysin) with or without 5 mM glycine treatment. Cytotoxicity was evaluated by measuring the levels of LDH in the supernatant. Cytotoxicity decreased in glycine-treated wildtype cells comparably to NINJ1 knockout across each of (B) pyroptosis, (C) post-apoptosis lysis, and (D) necrosis. Glycine treatment of NINJ1 KO cells provided no additional protection to knockout cells treated without glycine. Data are expressed as supernatant LDH as a % of total LDH from lysates and supernatants, from n = 3 independent experiments for each cell death type. Individual data points are shown along with their mean. * p<0.05 by ANOVA with Tukey’s multiple comparison correction. (E) LPS-primed wildtype iBMDM induced to undergo pyroptosis in the presence of glycine demonstrate similar plasma membrane ballooning to NINJ1 knockout iBMDM induced to undergo pyroptosis. Membrane ballooning is shown in live cells labeled with the plasma membrane dye FM4-64. The corresponding brightfield image is shown in the inset. Scale bar 15 µm.

Figure 1—source data 1. Numerical values for experimental data plotted in Figure 1.
elife-78609-fig1-data1.xlsx (651.4KB, xlsx)

Figure 1.

Figure 1—figure supplement 1. Glycine dose-dependently inhibits rupture of pyroptotic mouse macrophages with greater potency than structurally similar amino acids through inhibition of NINJ1 clustering.

Figure 1—figure supplement 1.

Primary LPS-primed mouse bone marrow-derived macrophage (BMDM) were treated for 90 min with increasing concentrations of glycine or the indicated concentrations of alanine, serine, or valine prior to induction of pyroptosis with nigericin as described in the main text. Supernatant LDH levels were measured 30 min later as a marker of cytotoxicity. (A) Glycine dose-dependently inhibits rupture of pyroptotic mouse BMDM with an IC50 between 1 and 2 mM, consistent with the published literature (Loomis et al., 2019). Experiment done in duplicate. (B) Glycine confers greater cytoprotection compared with alanine >> serine, valine as measured by LDH release. (C) BN-PAGE for NINJ1 shows that amino acid treatment limits NINJ1 aggregation in pyroptosis with a similar hierarchy of glycine > alanine > serine, valine.
Figure 1—figure supplement 1—source data 1. Numerical values for experimental data plotted in Figure 1—figure supplement 1.
Figure 1—figure supplement 2. Glycine cytoprotection phenocopies NINJ1 knockout and prevents NINJ1 aggregation in a mouse macrophage cell line.

Figure 1—figure supplement 2.

Using a CRISPR-Cas9 system, Ninj1 was knocked out of a RAW mouse macrophage cell line engineered to constitutively express ASC (RAW-ASC) and therefore have the capacity to undergo pyroptosis. (A) Western blot demonstrating NINJ1 knockout compared with the parental line. GAPDH is shown as a loading control. (B) Wildtype and NINJ1 knockout cells were induced to undergo pyroptosis with LPS + nigericin treatment with or without glycine. Supernatant LDH was evaluated as a marker of cytotoxicity by colorimetric assay. Cytotoxicity was decreased in glycine-treated (5 mM) wildtype cells comparably to NINJ1 knockout. Glycine (5 mM) treatment of NINJ1 KO cells provided no additional protection to knockout cells without glycine. * p<0.05 by two-sided ANOVA with Tukey’s multiple comparison correction. (C) LPS-primed wildtype RAW-ASC induced to undergo pyroptosis in the presence of glycine demonstrate similar plasma membrane ballooning to NINJ1 knockout RAW-ASC cells induced to undergo pyroptosis. Membrane ballooning is shown in live cells labeled with the plasma membrane dye FM4-64. Scale bar 20 µm. (D) Native-PAGE analysis of endogenous NINJ1 in RAW-ASC macrophages demonstrates a shift to high molecular weight aggregate upon pyroptosis stimulation, which is abrogated by glycine treatment.
Figure 1—figure supplement 2—source data 1. Numerical values for experimental data plotted in Figure 1—figure supplement 2.