1. Volume 25, Issue 4, Pages 447-459.e4 (April 2018)
N-Acetyl Cysteine Functions as a Fast-Acting Antioxidant by Triggering Intracellular H2S and Sulfane Sulfur Production 
Daria Ezeriņa, Yoko Takano, Kenjiro Hanaoka, Yasuteru Urano, Tobias P. Dick 
Cell Chemical Biology 
Volume 25, Issue 4, Pages e4 (April 2018)
DOI: /j.chembiol
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2. Cell Chemical Biology 2018 25, 447-459. e4DOI: (10. 1016/j. chembiol
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3. Figure 1
NAC Catabolism Induces Rapid Oxidation of Mitochondrial, but Not Cytosolic, roGFP2
(A) Treatment of cells with NAC triggers partial oxidation of mitochondrially localized roGFP2, but not of cytosolic roGFP2.
(B–D) The same phenomenon can be provoked more strongly by treating with Cys (B), 3MP (C), and the H2S donor Na2S (D).
(E) These findings led to the working hypothesis that NAC, Cys, 3MP, and H2S trigger roGFP2 oxidation through production of mitochondrial hydropersulfides.
(F) Exogenous application of the sulfane sulfur compound DMTS, which leads to the intracellular formation of persulfides, triggers roGFP2 oxidation in both compartments.
All curves represent the mean of three biological repeats, each carried out in technical quadruplicates, the error bars denoting the SD. OxD, degree of probe oxidation. The arrow indicates the time point of addition of the compound.
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4. Figure 2
NAC, Cys, and 3MP Trigger the Generation of H2S and Persulfides
(A) NAC, Cys, and 3MP trigger endogenous H2S generation, as detected by the probe HSip-1 DA.
(B) The mt-roGFP2 response is not significantly influenced by the translational fusion to Orp1, confirming that the observed response is H2O2 independent.
(C) The boronate-based mitochondrially targeted chemical H2O2 probe mitoPY1 does not detect increased H2O2 generation in response to NAC, Cys, 3MP, Na2S, or Na2S4, but rather a decrease. The last panel shows the response to H2O2 (positive control). The penultimate panel is also part of Figure 5E.
(D) NAC, Cys, 3MP, and H2S trigger endogenous sulfane sulfur generation, as detected by the sulfane sulfur probe SSip-1 DA.
All curves represent the mean of three biological repeats, each carried out in technical quadruplicates, the error bars denoting the SD. OxD, degree of probe oxidation. The arrow indicates the time point of addition of the compound.
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5. Figure 3 NAC-Induced H2S Production Involves CSE and MST
(A) N-Propargyl glycine (NPG) partially inhibits NAC- and Cys-induced mt-roGFP2 oxidation, but not 3MP- or Na2S-induced mt-roGFP2 oxidation.
(B) Genetic disruption of CSE expression partially inhibits NAC- and Cys-induced mt-roGFP2 oxidation, but not 3MP- or Na2S-induced mt-roGFP2 oxidation.
(C) Pyruvate partially inhibits NAC-, Cys-, and 3MP-induced mt-roGFP2 oxidation, but not Na2S-induced mt-roGFP2 oxidation.
(D) Genetic disruption of MST expression partially inhibits NAC-, Cys-, and 3MP-induced mt-roGFP2 oxidation, but not Na2S-induced mt-roGFP2 oxidation.
(E) Pyruvate treatment of CSE KO cells leads to a near-complete suppression of Cys-induced mt-roGFP2 oxidation, while Na2S-induced mt-roGFP2 oxidation is not significantly affected.
All curves represent the mean of three biological repeats, each carried out in technical quadruplicates, the error bars denoting the SD. OxD, degree of probe oxidation. The arrow indicates the time point of addition of the compound.
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6. Figure 4 NAC-Induced Persulfide Generation Involves SQR and MST
(A) NAC, Cys, and Na2S treatment leads to increased cellular oxygen consumption.
(B) Disruption of SQR expression suppresses NAC-, Cys-, and 3MP-induced sulfane sulfur generation, and completely abolishes H2S-induced sulfane sulfur generation, as detected by SSip-1 DA.
(C) Disruption of SQR expression suppresses NAC-, Cys-, and 3MP-induced mt-roGFP2 oxidation, and completely abolishes H2S-induced mt-roGFP2 oxidation, but does not influence DMTS-induced mt-roGFP2 oxidation.
All curves represent the mean of three biological repeats, each carried out in technical quadruplicates, the error bars denoting the SD. OxD, degree of probe oxidation. The arrow indicates the time point of addition of the compound.
Cell Chemical Biology  , e4DOI: ( /j.chembiol )
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7. Figure 5
NAC-Induced Generation of Persulfides May Provide Protection Against Oxidative Stress
(A) The cytoprotective effect of NAC against acute peroxide stress is mimicked by Cys, 3MP, and Na2S.
(B) Exogenous provisioning of Na2S4 provides protection against acute peroxide stress within a certain concentration range (around 100 μM), but not at higher concentrations (1 mM).
(C and D) The oxidation of the H2O2 probe roGFP2-Orp1 is suppressed by exogenously applied Na2S4 when applied at low concentrations (C). A very similar effect is observed with mitochondrial roGFP2-Orp1 (D).
(E) The response of the chemical H2O2 probe MitoPY1 is suppressed by exogenously applied Na2S4 in a concentration dependent manner.
All curves represent the mean of three biological repeats, each carried out in technical quadruplicates, the error bars denoting the SD. OxD, degree of probe oxidation. The arrow indicates the time point of addition of the compound.
Cell Chemical Biology  , e4DOI: ( /j.chembiol )
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