Fig. 1: Cyanide is endogenously produced in mouse tissues and human cells.

a–d, Cyanide production rates from tissue homogenates in homogenization medium containing 0.4 mM glycine (vehicle) or in medium supplemented with 10 mM glycine (Gly) were determined by the electrochemical (ECh) method after alkalinization. a, Comparison of cyanide generation from various tissues (at least n = 6 per group, biological replicates). b, Comparison of cyanide generation from male-versus-female mice (n = 6 per group, biological replicates). c, Detection of cyanide generation from liver homogenates using the Cyanalyzer LC–MS/MS method (n = 5 per group, biological replicates). d, Detection of cyanide generation from liver homogenates obtained from male mice using the spectrophotometric method (n = 5 per group, biological replicates). e, Treatment with HCN scavengers THC or CoE (10 µM), lowered the cyanide generation (ECh method) from mouse liver homogenates (n = 6 per group, biological replicates). f, Heat inactivation of proteins (HI), physical inactivation of proteins by multiple cycles of freezing and thawing (F&T) or SDS-induced protein denaturation (SDS) lowered the cyanide signal (ECh method) from mouse liver homogenates, compared to the control (CTR; at least n = 5 per group, biological replicates). g,h, Intracellular visualization (g) and quantification (h) of cyanide by confocal microscopy. Quantification of cyanide-specific signal using corrected total cell fluorescence (CTCF) values using two different cyanide-sensitive fluoroprobes Chemosensor P (CP) and a spiropyrane derivative of cyanobiphenyl (CSP) in human primary hepatocytes (n = 4 per group, biological replicates) and a human hepatoma line (HepG2; n = 6 per group or n = 5 per group, biological replicates, using CP or CSP probes, respectively) treated with a vehicle, 10 mM glycine (Gly) or 10 µM THC. Created with BioRender.com. i, Cyanide production in primary mouse and human hepatocytes and HepG2 cells treated with vehicle in standard medium containing 0.4 mM glycine (vehicle), addition of 10 mM glycine (Gly) or addition of 10 µM THC in control medium (ECh method; n = 4 per group biological replicates for primary human hepatocytes, n = 6 per group biological replicates for HepG2 cells). j, Effect of THC or CoE (10 µM) on the cyanide signal in HepG2 cells (ECh method; at least n = 6 per group, biological replicates). k, Cyanide production in a panel of mammalian cell lines in normal medium containing 0.4 mM glycine (vehicle), in medium supplemented with 10 mM glycine (Gly) or in −Ser/Gly medium for 24 h (ECh method; at least n = 5 per group, biological replicates). l, Cyanide production from human PBMCs and human neutrophils under basal conditions and after incubation with 10 mM glycine (Gly) for 4 h (ECh method; n = 6 per group, biological replicates). m, Cyanide production in HepG2 cells grown for 24 h in normal medium (containing 0.4 mM glycine) in the absence or presence of 100 µM SHMT inhibitor or in −Ser/Gly medium supplemented with 0.4–10 mM glycine (ECh method; at least n = 7 per group, biological replicates). n, Glycine levels in HepG2 cells under baseline conditions, after pharmacological inhibition of SHMT (iSHMT), after addition of 10 mM glycine to the culture medium or in −Ser/Gly medium for 24 h (ECh method; at least n = 5 per group, biological replicates). Data in a–f and h–n are expressed as the mean ± s.e.m. Data in a, b, e, f and h–n were analysed with a two-way analysis of variance (ANOVA) followed by Bonferroni’s multiple-comparisons test. Data in c, d and l were analysed with a two-sided Student’s t-test. *P < 0.05 and **P < 0.01 indicate significant differences.

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