Fig 2: ROS-induced oxidation promotes DR6 endocytosis.HeLa cells were treated with DM-αKG (15 mM) for 6 h to observe protein localization under confocal microscope and analyze protein amounts by fractionation, or 24 h to assess DR6 oxidation level and pyroptotic features (including cell morphology, GSDMC cleavage, and LDH release), unless specially defined. Inhibitors Trolox (400 μM), Genistein (20 μM) or MβCD (1 mM) was used to pretreat cells for 2 h. a, b Indication of DR6 puncta in cells (a) and DR6 expression in plasma membrane (PM) (b) in response to DM-αKG stimulation. DR6-GFP was transfected into cells, the cells were then pretreated with Trolox, followed by DM-αKG treatment. Endogenous Rab5a was used as an early endosome marker (a). The amounts of DR6 in PM were quantified and shown in b. c, d Effect of DR6 oxidation on DM-αKG-induced DR6 puncta in cells (c) and DR6 expression in PM (d). siRNA-resistant DR6WT and DR65CS were expressed in DR6 knockdown cells. e, f Effects of Genistein and MβCD on DM-αKG-induced DR6 puncta in cells (e) and DR6 expression in PM (f). DR6-GFP was transfected into cells, the cells were then pretreated with inhibitors, followed by DM-αKG treatment. g–i Effects of Genistein and MβCD on DM-αKG-induced caspase-8 activation (g), GSDMC cleavage and LDH release (h), and pyroptotic morphology (i). Cells were pretreated with inhibitors as indicated, followed by DM-αKG treatment. Tubulin was used to determine the amount of loading proteins. All data are presented as means ± SEM of two or three independent experiments. ***P < 0.001. The data were analyzed using two-way ANOVA followed by the Bonferroni test.

Fig 3: A working model of α-KG-induced pyroptosis.Under acidic environment, the metabolic enzyme MDH1 promiscuously catalyzes the conversion of α-KG to L-2HG, which then boosts ROS level. This signal is responded by the plasma membrane-localized death receptor DR6 by inducing the oxidation of DR6. Oxidized DR6 internalizes into the cytosol, and then recruits both pro-caspase-8 and GSDMC to the DR6 receptosome, in which active caspase-8 cleaves GSDMC, thereby inducing pyroptosis. This story demonstrates a novel pathway from ROS-initiated DR6 endocytosis to caspase-8-cleaved GSDMC for pyroptosis induction.

Fig 4: DR6 responds to ROS signals to induce pyroptosis.HeLa cells were treated with DM-αKG (15 mM) for 6 h to determine the ROS level or 24 h to assess the DR6 oxidation, caspase-8 activation, and pyroptotic features (including cell morphology, GSDMC cleavage, LDH release, and Annv+/PI+ cells), unless specially defined. Inhibitors Trolox (400 μM) and Z-VAD (40 μM) were used to pretreat cells for 2 h. a Determination of ROS levels at the indicated concentrations of DM-αKG (left), with or without pretreatment of Trolox (right). b Effect of Trolox on DM-αKG-induced activation of caspase-8. Active caspase-8 indicated by arrow was at p43 site. c, d Effects of DR6 on DM-αKG-induced pyroptotic morphology, GSDMC cleavage, LDH release (c) and Annv+/PI+ cell population (d). DR6 was knocked down first in HeLa cells, the cells were then treated with DM-αKG. e Effects of Trolox and Z-VAD or caspase-8 and GSDMC on DM-αKG-induced DR6 oxidation. In first two panels, Trolox or Z-VAD was used to pretreat cells. In last two panels, Caspase-8 or GSDMC was knocked down first in cells, the cells were then treated with DM-αKG. DR6 oxidation was observed by running non-reducing gels. f–h Effects of DR6 oxidation mutant DR65CS on DM-αKG-induced DR6 oxidation (f), caspase-8 activation (g) and pyroptotic characteristics (h). siRNA-resistant DR65CS was expressed in DR6 knockdown cells. DR6WT was used as a positive control. The expression levels of DR6WT-HA and DR65CS-HA were indicated in g (bottom). Tubulin was used to determine the amount of loading proteins. All data are presented as means ± SEM of two or three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. The data were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test (a (left), h) or two-way ANOVA followed by the Bonferroni test (a (right), c, d).

Fig 5: The acidic environment facilitates cell sensitivity to α-KG-induced pyroptosis.Different cancer cell lines, namely, U251, SK-MEL-1, MCF7, and MDA-MB-231 were treated with DM-αKG (15 mM) for 6 h to determine the L-2HG and ROS levels or 24 h to assess DR6 oxidation, caspase-8 activation, and pyroptotic features (including cell morphology, GSDMC cleavage, and LDH release), unless specially defined. a–e Effects of different pH on DM-αKG-induced GSDMC cleavage (a), L-2HG levels (b), ROS levels (c), DR6 oxidation (d), and caspase-8 activation (e). Different cancer cells were cultured in medium with different pH values as indicated. f–h Effects of lactic acid on DM-αKG-induced L-2HG levels (f), ROS levels (g), and GSDMC cleavage (h). Lactic acid (20 mM) together with DM-αKG were used to treat different cancer cells as indicated. i, j SK-MEL-1 cells were injected into nude mice to form subcutaneous xenografts (n = 6). Lactic acid (15 mg/kg) or DM-αKG (500 mg/kg) was intratumorally injected alone or combinedly every other day (four times). The xenograft tumors, tumor weights (i, n = 6) and GSDMC cleavage (j, n = 3) were indicated. Tubulin was used to determine the amount of loading proteins. All data are presented as means ± SEM of two or three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. The data were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test in i or two-way ANOVA followed by the Bonferroni test in b, c, f, g.