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. Author manuscript; available in PMC: 2021 Mar 1.

Published in final edited form as: Nat Chem Biol. 2020 Feb 17;16(3):278–290. doi: 10.1038/s41589-019-0462-8

Fig. 3. — (a) NO• donors (SNAP and DPTA NONOate) inhibit RSL3-induced ferroptosis in RAW 264.7 macrophages. Cells were treated with RSL3 (500 nM) in the presence of different concentrations of NO•-donors for 5 h. Cell death was estimated by LDH release assay. Fer-1 (400 nM) was used as a positive control. Data are mean ± s.d.; For RAW 264.7 macrophages, number of biologically independent samples in: control (n =4), RSL3 (n=10), RSL3+Fer-1 (n=6), DPTA-NONOate (n=6, 8, 8) for 5 μM, 12.5 μM and 25 μM, respectively, SNAP (n=4, 8, 6) for 5 μM, 12.5 μM and 25 μM, respectively. ^$p < 0.0001 vs control, *p < 0.0001 vs RSL3, ^#p = 0.0004 vs RSL3, two-way ANOVA, Tukey’s multiple comparisons test.

(b) NO• protects RAW 264.7 macrophages against ferroptosis induced by ML162, IKE or erastin. Cells were treated with ML162 (2 μM), IKE (2 μM) or erastin (20 μM) in the presence of 25 μM of DETA NONOate for 18 h. Cell death was estimated by LDH release assay. Fer-1 (400 nM) was used as a positive control. Data are means ± s.d.; Number of biologically independent samples in control (n=9), ML-162( n=6, 6, 9) IKE (n=9, 8, 7) erastin (n=9, 9, 8) for inducer of ferroptosis, RSL3, RSL3+Fer-1 groups, respectively. ^$p < 0.0001 vs control, *p < 0.0001 vs ferroptosis inducer, ^#p < 0.0001 vs ferroptosis inucer, two-way ANOVA, Tukey’s multiple comparisons test.

(c) NO• donors (SNAP and DPTA NONOate) inhibit RSL3-induced ferroptosis in MLE-cells. Cells were treated with RSL3 (500 nM) in the presence of different concentrations of NO•-donors for 5 h. Cell death was estimated by LDH release assay. Fer-1 (400 nM) was used as a positive control. Data are mean ± s.d.; n=4 biologically independent samples per group, ^$p < 0.0001 vs control, *p < 0.0001 vs RSL3, two-way ANOVA, Tukey’s multiple comparisons test.

(d) NO• released by iNOS overexpressing M1 activated RAW 264.7 (but not M2 alternatively activated macrophages) inhibit RSL3-induced ferroptosis in MLE cells. MLE cells labeled with Cell Tracker Green were incubated with or without unlabeled M1 or M2 RAW 264.7 macrophages (macrophages to MLE ratio, 2:1). Ferroptosis was induced by treatment with RSL3 (500 nM) for 5 h in the absence or in the presence of Fer-1 (500 nM). Cell death was estimated by PI-staining using flow cytometer and calculated as percentage of double positive cells (Cell Tracker Green and PI) to total amount of Cell Tracker Green-positive cells. Data are means ± s.d.; Number of biologically independent samples: control=11, RSL3+Fer-1=3, RSL3=10, 9, 4 for MLE, MLE+M1 and MLE+M2, respectively, RSL3+L-NIL=4, 3 for MLE+M1 and MLE+M2, respectively, 1400W =7. ^$p < 0.0001 vs control, *p < 0.0001 vs RSL3, ^#p < 0.0001 vs MLE cells treated with RSL3 and incubated with M1 macrophages, two-way ANOVA, Tukey’s multiple comparisons test.

(e) GPX4 knock down increased sensitivity to RSL3-induced ferroptosis in non-activated (M0) and alternatively activated (M2) RAW 264.7 macrophages but not in M1 macrophages. (1) Densitometry-based assessments of mean relative intensity for the protein demonstrating efficiency of GPX4 knock-down. Data are means ± s.d.; n=3 biologically independent samples. *p = 0.0311 vs M0/Control, ^$p = 0.0023 vs. M0/si-NT, **p = 0.0328 vs M1/Control, ^$$p = 0.0168 vs. M1/si-NT, ***p = 0.0432 M2/Control and ^$$$p = 0.0165 vs. M2/si-NT, two-way ANOVA, Tukey’s multiple comparisons test.

(f) NO• donors prevented ferroptosis induced in human bronchial epithelial (HBE) cells by Pseudomonas aeruginosa supernatants. HBE cells were incubated with P. aeruginosa supernatant (Sup) alone or with two different types of NO• donors (SNAP or DETA NONOate) for 20 h. Cell death was estimated by PI-staining using flow cytometry. Fer-1 was used as a positive control. Data are means ± s.d., n = 3 biologically independent samples, ^$p = 0.0125 vs control, ^$$p = 0.0162 vs Sup., *p = 0.0435 vs Sup., **p = 0.0039 vs Sup., ^#p = 0.0154 vs Sup., one-way ANOVA, Tukey’s multiple comparisons test.

(g) Pre-treatment of RAW 264.7 macrophages with P. aeruginosa supernatant induced iNOS expression, depleted GPX4 and rendered macrophages resistant to ferroptosis. RAW 264. Macrophages were pre-treated with P. aeruginosa supernatants (Sup) overnight and then ferroptosis was triggered by addition of RSL3 (500 nM) for 5 hrs. Fer-1 was used as a positive control. Data are means ± s.d.; Number of biologically independent samples: control (n=3 each), RSL3/-Sup (n=5), RSL3/+Sup (n=6) , RSL3/Fer-1 (n=5). ^$p < 0.0001 vs control/-Sup and control/+Sup, *p = 0.0011 vs. RSL3/-Sup., **p < 0.0001 vs. RSL3/-Sup., two-way ANOVA, Tukey’s multiple comparisons test. Inset: Representative Western blots of iNOS and GPX4.