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. 2022 Dec 5;11:e78609. doi: 10.7554/eLife.78609

Figure 2. NINJ1 silencing in primary human monocyte-derived macrophages (hMDMs) functionally and morphologically phenocopies glycine cytoprotection.

Primary hMDMs were treated with siRNA targeting NINJ1 (siNINJ1) or non-targeted control (siCtrl). (A) Wildtype and NINJ1-silenced hMDMs were LPS-primed and induced to undergo pyroptosis (nigericin) with or without 50 mM glycine. LDH release in the supernatants was measured to assess cytotoxicity. Cytotoxicity was reduced in glycine-treated wildtype cells, as well as in NINJ1-silenced cells. Glycine treatment did not yield additional protection from pyroptotic cell death in NINJ1-silenced cells. Data are expressed as % of LDH in supernatant from siCtrl-treated cells stimulated to undergo pyroptosis from n=4 independent donors. Data points from each donor are shown along with their mean and SD. **** p<0.0001 by two-way ANOVA with Tukey’s multiple comparison correction. (B) Nigericin- and glycine-treated LPS-primed wildtype hMDMs show similar ballooning morphology as nigericin-treated LPS-primed NINJ1-silenced hMDMs. Membrane ballooning is shown in live cells labeled with the plasma membrane dye FM1-43. The corresponding brightfield image is shown in the inset. Scale bar 20 μm.

Figure 2—source data 1. Numerical values for experimental data plotted in Figure 2.

Figure 2.

Figure 2—figure supplement 1. Glycine dose-dependently inhibits rupture of pyroptotic human monocyte-derived macrophages (hMDMs).

Figure 2—figure supplement 1.

Primary human macrophages were pre-incubated with increasing doses of glycine and primed with LPS (1 µg/mL) for 3 hr before addition of nigericin (20 µM). Levels of LDH and IL-1β in supernatants were measured 2 hr later as described in the main text. Experiment done in triplicate.
Figure 2—figure supplement 1—source data 1. Numerical values for experimental data plotted in Figure 2—figure supplement 1.
Figure 2—figure supplement 2. Glycine does not confer cytoprotection in necroptotic human macrophages.

Figure 2—figure supplement 2.

Primary human macrophages pre-treated or not with glycine (50 mM), pan-caspase inhibitor zVAD (25 µM), and SMAC mimetic BV6 (10 µM) for 30 min before stimulation with TNF (10 ng/mL). Supernatant LDH levels were measured 4 hr later as a measure of cytotoxicity. **** p<0.0001, *** p<0.001 by two-way ANOVA with Tukey’s multiple comparison correction. ns = not significant.
Figure 2—figure supplement 2—source data 1. Numerical values for experimental data plotted in Figure 2—figure supplement 2.
Figure 2—figure supplement 3. Glycine preserves membrane integrity but not cell viability in human macrophages undergoing pyroptosis.

Figure 2—figure supplement 3.

Human monocyte-derived macrophages (hMDMs) were either left untreated or pre-treated with glycine (50 mM) and primed with 1 μg/mL LPS before stimulation with nigericin (20.7 µM). To assay cell viability, the mitochondrial membrane potential sensitive dye tetramethylrhodamine ethyl ester (TMRE) perchlorate was added, and images were taken every 30 min overnight. (A) Representative images of TMRE staining (green) from two independent experiments where the second includes CellTracker Deep Red dye as a cellular marker (red, lower panel). Scale bars 100 µm. (B) TMRE intensity decreases, and cells die prior to ballooning in cells undergoing pyroptosis (30 min between images shown, except last image which shows the same cell at 18 hr). (C) Frequency of TMRE positive cells, (D) LDH release, and (E) ATP content at indicated time points. Results from two to three independent experiments are shown in (C–E) with different symbols for individual donors.
Figure 2—figure supplement 3—source data 1. Numerical values for experimental data plotted in Figure 2—figure supplement 3.