Extended Data Fig. 4: Mechanisms of lysosomal HCN production.
From: Regulation of mammalian cellular metabolism by endogenous cyanide production
(a) Western blot of cytosolic (C) vs lysosomal (L) fractions from HepG2 cells. LAMP-1: lysosomal associated membrane protein 1; GAPDH: glyceraldehyde 3-phosphate dehydrogenase. (b) Western blot of lysosomal (L), extra-lysosomal (EL) and cytosolic (C) fractions from mouse liver. (c) HCN detection in the lysosomal vs cytosolic fractions of HepG2 cells, with or without incubation with glycine (10 mM), as measured with the LC-MS/MS method (at least n = 4, biological replicates). (d) Inhibitory effect of the lysosomal proton pump inhibitor bafilomycin (Baf, 0.1- 1 µM) on cyanide production in HepG2 cells in the presence of increasing concentration (10 mM) of glycine (Gly) (at least n = 4/group, biological replicates). (e) Inhibitory effect of the lysosomal deacidifier hydroxychloroquine (1-100 µM) on cyanide production in HepG2 cells (at least n = 4/group, biological replicates). (f) Inhibitory effect of the peroxidase inhibitor phloroglucinol (Phl, 1- 30 µM) on cyanide production in HepG2 cells (at least n = 4/group, biological replicates). (g) Western blots of the expression of catalase and various peroxidases – catalase, peroxiredoxin3 (PRDX3), peroxiredoxin6 (PRDX6), glutathione S-transferase alpha1 (GSTA1), glutathione S-transferase alpha2 (GSTA2), and microsomal glutathione S-transferase1 (MGST1) – in cytosolic (C) vs. lysosomal (L) fractions of HepG2 cells, including densitometric quantification of their relative expression (n = 5/group, biological replicates). Data in c, d, e, f, g, are expressed as the mean ± s.e.m. Data in c, d, e, f were analysed with a two-way ANOVA followed by Bonferroni’s multiple-comparisons test. Data in g were analysed with a two-sided Student’s t-test. *p < 0.05 and **p < 0.01 indicate significant differences.
