Extended Data Fig. 7: Endogenous HCN modulates cellular bioenergetics in HepG2 cells: extracellular flux analysis.
From: Regulation of mammalian cellular metabolism by endogenous cyanide production
(a) Glycine-dependent stimulation of cellular bioenergetics is abrogated by HCN scavengers. Extracellular flux analysis of HepG2 cells incubated with 10 mM glycine (Gly) for 24 h followed by 3 h with 10 µM of the cyanide scavengers (trihistidyl-cobinamide (THC) or dicobalt edetate (CoE). OCR: oxygen consumption rate (at least n = 5/group, biological replicates). (b) HepG2 cells incubated with serine/glycine free medium (-Ser/Gly) or with serine/glycine free medium re-supplemented with 0.4 mM serine and 0.4 mM glycine (+Ser/Gly) (at least n = 5/group, biological replicates). (c,d). Glycine-dependent stimulation of cellular bioenergetics is abrogated by overexpression of the HCN-catabolizing enzyme cyanide dihydratase (CynD) in HepG2 cells, compared to normal control wild-type (WT) cells. Extracellular flux analysis (n = 6/group, biological replicates). (e,f) Free fatty acid oxidation extracellular flux analysis of (e) control HepG2 cells and (f) HepG2 cells pretreated with 4 µM etomoxir (carnitine palmitoyl transferase-1 inhibitor). In both cases, cells were pre-incubated in presence of 10 mM glycine or vehicle for 24 h. Arrows in panels a, b, c, e and f represent the times of the addition of the ATP synthase inhibitor oligomycin, the uncoupling agent carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) and the combined addition of the mitochondrial Complex I inhibitor rotenone and the mitochondrial Complex III inhibitor antimycin (R/A) in the Extracellular Flux Analysis protocol (at least n = 5/group, biological replicates). Data are expressed as the mean ± s.e.m. Data were analysed with a two-way ANOVA followed by Bonferroni’s multiple-comparisons test. *p < 0.05 and **p < 0.01 indicate significant differences.
