Fig 2: Iron acts as a sensitizer for different drugs and induces pyroptosis in melanoma cells. Melanoma A375 cells were treated with SSZ (sulfasalazine, 125 μM) with or without FeSO4 (100 μM) for 6 h to detect the ROS level or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release), unless specifically defined. a FeSO4 acts as a sensitizer for ROS generation, GSDME cleavage, LDH release, and pyroptosis in the presence of SSZ. b, c Extensive treatment with SSZ/FeSO4 at the indicated times induced Tom20 oxidation and accumulation (b) and GSDME cleavage (c). d–g Separate knockdown of Tom20, Bax, caspase-3 or GSDME blocked SSZ/FeSO4-induced pyroptosis, GSDME cleavage, and LDH release as indicated. Tubulin was used to determine the amount of loading proteins. All data are presented as the mean ± SEM of three independent experiments. **P < 0.01, ***P < 0.001

Fig 3: Tom20-induced translocation of Bax to mitochondria contributes to pyroptosis. Melanoma A375 cells were treated with CCCP (20 μM), FeSO4 (100 μM), or CCCP/FeSO4 for 6 h to detect the translocation of Bax to mitochondria, cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release) and cell death, unless specifically defined. To detect the effects of the Tom20 point mutants Tom20C13S and Tom20C21S, Tom20 was knocked down in A375 cells, and Tom20WT or its point mutants Tom20C13S and Tom20C21S were separately transfected into cells. a Bax translocated to mitochondria upon CCCP/FeSO4 stimulation as shown by confocal microscopy. b Knockdown of Tom20 blocked the translocation of Bax to mitochondria upon CCCP/FeSO4 treatment. The mitochondrial fraction in cells was prepared. c Knockdown of Bax blocked the CCCP/FeSO4-induced mitochondrial aggregation. d-g After knocking down Bax, the CCCP/FeSO4-induced cytochrome c release detected in the cytosol fraction was diminished (d), the cleavage of caspase-3 and -9 was attenuated (e), the cell viability was rescued (f), the cell morphologies were reversed from pyroptosis to normal state, and LDH release and GSDME cleavage were also abolished (g). h Effects of the mutants Tom20C13S and Tom20C21S on the translocation of Bax to mitochondria in response to CCCP/FeSO4 stimulation. Hsp60 was used to detect the mitochondria, and DAPI was used to display the nuclei by confocal microscopy. Tubulin was used to determine the amount of loading proteins. Hsp60 was used to determine the amount of mitochondrial proteins. All data are presented as the mean ± SEM of three independent experiments. ***P < 0.001

Fig 4: Mitochondrial pathway with activation of caspase-3 is involved in pyroptosis induced by iron. Melanoma A375 cells were pretreated with or without different inhibitors, including NAC (5 mM) or GSH (1 mM) for 2 h, followed by CCCP (20 μM), FeSO4 (100 μM), or CCCP/FeSO4 treatment for 6 h to detect cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release), unless specifically defined. a Mitochondrial depletion blocked CCCP/FeSO4-induced pyroptosis, GSDME cleavage and LDH release. Mito: mitochondria. b CCCP/FeSO4 induced mitochondrial accumulation, but NAC and GSH attenuated this accumulation. c CCCP/FeSO4 induced cytochrome c release from mitochondria to cytosol as detected in the cytosol fraction (left) or by confocal microscopy (right). Cyto C: cytochrome c. d, e NAC or GSH abolished CCCP/FeSO4-induced cytochrome c release (d) and cleavage of caspase-3 and -9 (e). CASP: caspase. f Knockdown of FTL or FTH1 enhanced the CCCP/FeSO4-induced cleavage of caspase-3 and -9. g, h Knockdown of either caspase-3 or -9 abolished the CCCP/FeSO4-induced GSDME cleavage (g), pyroptosis and LDH release (h). Tubulin was used to determine the amount of loading proteins. All data are presented as the mean ± SEM of three independent experiments. **P < 0.01, ***P < 0.001

Fig 5: Iron-induced Tom20 accumulation promotes pyroptosis. Melanoma A375 cells were pretreated with or without different inhibitors, including NAC (5 mM) or GSH (1 mM) for 2 h, followed by CCCP (20 μM), FeSO4 (100 μM), or CCCP/FeSO4 treatment for 6 h to detect cytochrome c release and caspase-3 or -9 cleavage or 24 h to assess the pyroptotic features (including morphology, GSDME cleavage, and LDH release) and cell death, unless specifically defined. a Top, addition of FeSO4 to CCCP induced Tom20 and Tom40 accumulation. Bottom, the effect of CCCP/FeSO4 on the expression levels of Tom20 and Tom40. Cells were treated with CCCP or CCCP/FeSO4 for the indicated times. b Knockdown of Tom20 rescued the cell viability in response to CCCP/FeSO4 stimulation. c–e Knockdown of Tom20 reversed the CCCP/FeSO4-induced cell morphology from pyroptosis to normal state, reduced LDH release and blocked GSDME cleavage (c), abolished cytochrome c release (detected in the cytosol fraction) and cleavage of caspase-3 and -9 (d), and blocked mitochondria aggregation (e). Hsp60 was used as a mitochondrial indicator, and DAPI was used to display the nuclei. f NAC and GSH attenuated the CCCP/FeSO4-induced Tom20 accumulation. Tubulin was used to determine the amount of loading proteins. Hsp60 was used to determine the amount of mitochondrial proteins. All data are presented as the mean ± SEM of three independent experiments. ***P < 0.001