Fig. 5.

Glycine-based treatment decreases superoxide generation in macrophages through GSH formation. Bone marrow-derived macrophages (BMDMs) were treated with glycine-depleted metabolic media supplemented with water control, glycine (1 mM) or DT-109 (1 mM) for 18 h. (A) Following treatment, BMDMs were labeled with dihydroethidium (DHE, red) for assessment of superoxide and nuclei were stained using Hoechst (blue) (scale bar: 100 μm). (B) DHE fluorescence was measured as mean fluorescence intensity normalized to the number of nuclei per high powered field and expressed as fold change from water control (n = 3). (C) Schematic diagram illustrating the production of GSH by glutamate-cysteine ligase, with the addition of inhibitors (Gclm KO, glycine depletion) to enhance superoxide production (γ-GC, γ-glutamylcysteine). (D) BMDMs were isolated from Gclm^+/+ and Gclm^−/- mice, and the absence of GCLM was confirmed by Western blot using GAPDH as loading control (n = 3). (E) Following 18 h treatment with glycine-depleted media supplemented with water control, glycine (1 mM) or DT-109 (1 mM), superoxide was analyzed in BMDMs from Gclm^−/- mice using DHE (scale bar: 100 μm). (F) DHE fluorescence was measured as mean fluorescence intensity normalized to the number of nuclei per high powered field and expressed as fold change from water control (n = 4). Data are presented as mean ± SEM showing all points. Statistical differences were compared using one-way ANOVA with Tukey's post hoc analysis or Kruskal-Wallis test followed by Dunn's post-hoc analysis. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)