Extended Data Fig. 3: Confocal microscopy analysis of cyanide and HOCl generation in HepG2 cells.

(a) Workflow of HCN detection using confocal microscopy. MPO – myeloperoxidase. Created with BioRender.com. (b) Confocal microscopy of HepG2 cells incubated with 10 µM selective HCN probe for 1 h incubation together with other cell-permeant dyes (10 µM Calcein AM, 1 µM CellMask Green Actin Tracking Stain or 200 nM MitoTracker Deep Red FM) for 30 minutes at 37 °C and 5% CO₂. At the end of the incubation, cells were washed 3x and visualized using confocal microscope. Following excitation and emission spectra were used: HCN probe (Ex405/Em584-620 nm), Calcein AM (Ex488/Em517 nm), CellMask Plasma Membrane Stain (Ex488/Em535 nm) MitoTracker Deep Red FM (Ex644/Em665 nm). Images shown are representative of n = 3 biological replicates/group. (c) Co-localization of MPO with various subcellular organelles. Representative confocal microscopy images of HepG2 showing that MPO does not localize to the endoplasmic reticulum (ER) nor mitochondria. ER was visualized by using 500 nM ER tracker green. Primary anti-MPO antibody (mouse, dilution 1:10,000, Sigma-Aldrich) followed by incubation with appropriate secondary antibody goat anti-rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor Plus 647 (1:1,000 dilution) was used for detection of MPO. Mitochondria were visualized by using 200 nM MitoTracker Deep Red FM stain, while cellular nuclei were stained by 5 µg/ml DAPI. Images were collected at Leica 8 STELLARIS Falcon using 63x magnification. For co-localization with ER, ER tracker was visualized at Ex504/Em511 nm and MPO at Ex647/Em665 nm. For co-localization with mitochondria, MitoTracker was visualized at Ex647/Em665 nm and MPO at Ex568/Em603 nm. Images shown are representative of n = 3 biological replicates/group. (d) Co-localization of HOCl with lysosomes. Representative confocal microscopy image of HepG2 showing that HOCl localizes with lysosomes. HepG2 cells were loaded with 10 µM Chemosensor P in HBSS for 1 h together with 50 nM LysoTrackerGreen for 30 min at 37 °C and 5% CO₂. At the end of the incubation, cells were washed 3x and visualized using confocal microscope. Following excitation and emission spectra were used: HOCl probe (Ex405/Em450-550 nm) and LysoTrackerGreen Ex488/Em517 nm. Images shown are representative of n = 3 biological replicates/group.

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