(A) Schematic illustration of the labeling of Met in cells using click chemistry. (B) Fluorescence microscopy images of labeled Met (green). Cells were treated with Met and subjected to click-labeling as described in Methods. Mitochondria were detected using cytochrome c immunostaining (red) and 4',6-diamidino-2-phenylindole (DAPI) stains nuclear DNA (blue). Scale bar, 10 μm. (C) Fluorescence microscopy detection of Cu²⁺ in cancer cells treated as indicated for 48 h. Scale bar, 10 μm. (D) Flow cytometry analysis of mitochondrial Cu²⁺ in cancer cells treated as indicated for 48 h. Bar chart represents an average of three independent experiments. (E) Flow cytometry analysis of mitochondrial Cu⁺ in cancer cells treated as indicated for 48 h. Bar chart represents an average of three independent experiments. (F) Cyclic voltammetry measurements towards oxidation potentials (purple arrow) of a Cu⁺ solution. Data recorded in the absence (black) and presence of 2 mol equiv. metformin (blue) or 2 mol equiv. Met (red). Redox peak potentials are marked with dashed lines. (G) Flow cytometry analysis of Fe²⁺ in cancer cells treated as indicated for 48 h. MDA-MB-468 cells were used in Fig 2C, 2D, 2E and 2G and were treated as described in Methods. Cells were treated at the IC₅₀ concentration at 72 h of Metformin or Metforminyn respectively, unless stated otherwise. See also S6, S7, S8, S9, S10 and S11 Figs. Bar chart represents an average of three independent experiments.

https://doi.org/10.1371/journal.pone.0208213.g001