Fig. 3: HNO is generated by NO in the presence of reduced lipoate and is detectable in BMDMs.

a Test tube reactions were carried out in the presence of GSH and AS or mixtures of either oxidized or reduced-lipoate with DEA/NO for 1 h at 37 °C. GSH-sulfinamide was quantified by ESI-LC/MS-MS. Boxplots with floating bars (min to max) are shown with line at median. In (b) and (c) recombinant human DLD (rhDLD) was added to test tube reactions in the presence or absence of reduced lipoate and GSH-sulfinamide and sulfinic acid were quantified (n > 2 wells). Data were analyzed by one-way ANOVA with Dunnet’s multiple comparisons test. d WT BMDMs from male and female mice (mixed sex in all groups) were incubated with fluorescence probe for HNO for 30 min and stimulated with LPS + IFNγ for 4 h or with NOS2 inhibitor Aminoguanidine (AG) 1 h prior to stimulation. After PBS washing, fluorescence was measured in total cell lysates. For AS and DEA/NO conditions, cells were treated with donors for only 30 min. Data (n = 3 mice) were analyzed by one-way ANOVA with Dunnet’s multiple comparisons test. e Schematic illustration of mechanism of action of ML226 on ABHD11, which ensures stability of lipoate groups of PDH and OGDH enzymes. f Quantifications (percentage relative to LPS + IFNγ-stimulated cells) of fluorescence for HNO detection in cells treated with ML226 1 h prior to stimulation. Data (n = 3 mice) were analyzed by unpaired t test (two-tailed). All error bars display mean ± SEM. Data shown are representative of 2 or more independent experiments except (b) which is cumulative data from 2 independent experiments. Source data are provided as a Source Data file.