Fig 2: Endogenously generated persulfides scavenge APEX2-generated intracellular radicals.(A) Area under the luminescence curve obtained after pretreating APEX2 expressing HeLa cells with the indicated concentrations of Na2S (left panel) or 3MP (right panel) for 5 min before the addition of H2O2 (50 µM). n = 3. p = 0.0451. p = 0.0040. p = 0.0003. (B) Influence of MPST depletion on the luminescence recorded from APEX2-expressing HeLa cells after addition of H2O2 (50 µM) (left panel). Corresponding quantitation of the area under the luminescence curve (right panel). n = 3. p = 0.0198. (C) Influence of CSE overexpression (upper left panel), CSE depletion (upper middle panel), MPST depletion (upper right panel), ETHE1 overexpression (lower left panel), and ETHE1 depletion (lower right panel) on the area under the luminescence curve measured in HeLa cells expressing APEX2 in the mitochondrial matrix. n = 4. p = 0.00004. p = 0.00004. (CSE OE, siCSE); n = 3. p = 0.0435 (siMST); n = 4. p = 0.0474 (ETHE OE); n = 5. p = 0.0001 (siETHE1). Data are presented as mean values. Error bars represent SD, * P = 0.05; ** P = 0.01; *** P = 0.001; **** P = 0.0001 based on a two-tailed unpaired t-test. Source data

Fig 3: Influence of S0 metabolizing enzymes on membrane integrity.(A) Influence of CSE depletion on the viability of HeLa cells treated for 24 h with either RSL3 (5 µM) or CHP (40 µM), as measured with an ATP assay. n = 4. p = 0.00003 (RSL3), p = 0.00001 (CHP). (B) Influence of CSE overexpression (OE) on the loss of viability of U2OS cells treated with either RSL3 (5 µM) (left panels) or CHP (40 µM) (right panels), measured with the CellTox Green cytotoxicity assay. Rightmost panel: Influence of CSE overexpression (OE) on the viability of U2OS cells treated for 24 h with either RSL3 (5 µM) or CHP (40 µM), as measured with an ATP assay. EV: empty vector. n = 3. Right panel: n = 4, p = 0.2763 (RSL3); n = 5, p = 0.0029 (CHP). (C) Depletion of ETHE1 in HeLa cells, demonstrated by immunoblotting. (D) Influence of ETHE1 depletion on the loss of viability of HeLa cells treated with either RSL3 (5 µM) (left panels) or CHP (40 µM) (right panels), measured with the CellTox Green cytotoxicity assay. n = 3. (E) Overexpression of ETHE1 in HeLa cells, demonstrated by immunoblotting. (F) Influence of ETHE1 overexpression on the loss of viability of HeLa cells treated with either RSL3 (5 µM) (left panels) or CHP (40 µM) (right panels), measured with the CellTox Green cytotoxicity assay. n = 3. (G) Depletion of SQR in HeLa cells, as demonstrated by fluorescent antibody labeling. (H) Influence of SQR depletion on the loss of viability of HeLa cells treated with either RSL3 (5 µM) (left panels) or CHP (40 µM) (right panels), measured with the CellTox Green cytotoxicity assay. n = 3. Data are presented as mean values. For CellTox assay error bars represent SEM. For the rest, ** P = 0.01; **** P = 0.0001 based on a two-tailed unpaired t-test. Source data

Fig 4: Endogenously produced S0 eliminates intracellular radicals.a, Selected enzymatic pathways of hydropersulfide (RSSH) formation and elimination. CSE drives RSSH formation primarily through production of H2S, which is oxidized to RSSH by SQR. MPST provides an H2S-independent route to RSSH. Hydropersulfides are degraded by the persulfide dioxygenase ETHE1, which oxidizes S0 to sulfite (SO32-). b, Influence of Na2S (5 µM, substrate for SQR), 3MP (100 µM, substrate for MPST) or Cys (1 mM, substrate for CSE) on the radical load of APEX2-expressing HeLa cells, added 5 minutes before triggering radical generation with luminol, substrate and H2O2 (50 µM). n = 3. P = 0.0011, 0.0011 and 0.0136. c–f, Influence of CSE overexpression (c), CSE depletion (d), ETHE1 overexpression (e) and ETHE1 depletion (f) on the luminescence profiles recorded from APEX2-expressing HeLa cells (left panels). Cells were incubated with luminol and substrate (250 µM each), and radical generation was triggered with H2O2 (50 µM). Normalized AUC (right panels). EV, empty vector. n = 4. P = 0.0056 (c). n = 3. P = 0.0009 (d). n = 4. P = 0.0001 (e). n = 4. P = 0.0016 (f). Data are presented as mean values. Error bars represent s.d. * P = 0.05; ** P = 0.01; *** P = 0.001; and **** P = 0.0001 based on a two-tailed unpaired t-test. a.u., arbitrary units; OE, overexpressing.Source data

Fig 5: S0-generating/degrading enzymes modulate LPO and ferroptosis.a, Influence of depleting CSE (siCSE) on the loss membrane integrity triggered by treatment of HeLa cells with either RSL3 (5 µM) (left two panels) or CHP (40 µM) (right two panels), as measured by the CellTox Green cytotoxicity assay. n = 3. b, Influence of overexpressing CSE (CSE OE) on the loss of membrane integrity triggered by treatment of HeLa cells with either RSL3 (5 µM) (left two panels) or CHP (40 µM) (right two panels), as measured by the CellTox Green cytotoxicity assay. n = 3. c, Influence of depleting CSE (siCSE) or ETHE1 (siETHE1) on the loss membrane integrity triggered by treatment of HeLa cells with RSL3 (5 µM), as measured by the CellTox Green cytotoxicity assay. Fluorescence is normalized to the lysis control (lethal fraction). n = 3. d, Influence of depleting CSE (siCSE) on the oxidation of BODIPY-C11, as induced by incubation with RSL3 (5 µM) for 4 hours. n = 3. P = 0.0049. e, Influence of depleting ETHE1 (siETHE1) on the oxidation of BODIPY-C11, as induced by incubation with RSL3 (2 µM) for 3 hours. n = 3. P = 0.0236. Data are presented as mean values. For CellTox assays, error bars represent s.e.m. For the rest, error bars represent s.d. * P = 0.05 and ** P = 0.01 based on a two-tailed unpaired t-test.Source data