Fig 2: SIGLEC12 mutations and the proposed model of TMPRSS4-mediated SIGLEC12 activation during necroptosis.a, Summary of the SIGLEC12 mutation frequencies found in the general population (graphs) and their location in the SIGLEC12 domains. Green circles: extracellular mutations; Red circle: cancer-associated mutation S458F; Yellow circles: intracellular mutations; TM: transmembrane region. Blue boxes: V-type Ig domains; Green boxes: C2-type Ig domains; Orange boxes: ITIM and ITSM domains; Red box: NISI motif. b, A proposed model of SIGLEC12 activation and plasma membrane rupture induction during necroptosis. Panels a and b were created using Biorender (https://biorender.com).

Fig 3: The role of the NISI motif in PMR during necroptosis.a, HT-29-SIGLEC12-KO-SIGLEC12-FL-WT or NISI mutant (NISI-4A) stable cell lines were treated with TSE, and cell death was measured by green fluorescence intensity every hour for the indicated times using Incucyte S3 (p-value = <1 × 10−15, 5.99 × 10−9, 1.53 × 10−6). b, c, 293 T cells were co-transfected with indicated SIGLEC12-FL-FLAG plasmids ± TMPRSS4-HA for 40 h (b). 293FT-MLKLQ356A-SIGLEC12-KO cells were transfected with the indicated SIGLEC12-FL-FLAG plasmids and treated with 0.1 μg/ml doxycycline for 24 h to induce the expression of MLKL-Q356A (c). Cell death was measured using the Toxilight assay (p-value = 2.91 × 10−7 (b) and 8.26 × 10−8, 3.74 × 10−7 (c)). Data were plotted as the mean ± s.d., representative of n = 3 (a, left) or n = 9 (a, middle and right, b and c); three independent experiments. Statistical analyses were performed using two-tailed t-test and two-way ANOVA; ****p < 0.0001.

Fig 4: SIGLEC12 is cleaved by TMPRSS4 to induce plasma membrane rupture during necroptosis.a, Predicted structure of the [22-410aa]-truncated SIGLEC12 (SIGLEC12-ΔN). The red box shows the NINJ1/SIGLEC12 homology motif (NISI motif). b, 293 T cells were transfected with full-length (SIGLEC12-FL-FLAG) or [22-410aa]-truncated SIGLEC12 (SIGLEC12-ΔN-FLAG) for 40 h with or without NSA co-treatment. Cell death was measured using the Toxilight assay (p-value = 9.62 × 10−8). c, HeLa cells were transiently transfected with signal-peptide-HA-SIGLEC12-ΔN plasmid, and immunofluorescence was performed with or without cell permeabilization as indicated. Scale bars, 10 μm. d, HT-29 cells with shRNA-mediated stable knockdown of TMPRSS4 (two different shRNA sequences) were immunoblotted with the indicated antibodies following necroptosis induction by TSE. e, f, 293 T cells were co-transfected with indicated SIGLEC12-FLAG plasmids ± TMPRSS4-HA for 24 h. At 24 h post-transfection, cell lysates and anti-FLAG immunoprecipitation samples were immunoblotted with the indicated antibodies (e). Cell death was quantified at 40 h post-transfection by Toxilight assay (p-value = 9.69 × 10−7, 8.32 × 10−7) (f). g, HeLa-RIPK3-HA cells were transfected with indicated SIGLEC12-FLAG plasmids for 16 h and treated with TSE for 4 h. Cell lysates and anti-FLAG immunoprecipitation samples were analyzed by immunoblotting. h, i, 293 T cells were co-transfected with indicated SIGLEC12-FLAG plasmids ± TMPRSS4-HA for 40 h (h). 293FT-MLKLQ356A-SIGLEC12-KO cells were transfected with the indicated SIGLEC12-FLAG plasmids and treated with 0.1 μg/ml doxycycline for 24 h to induce MLKL-Q356A expression (i). Cell death was measured using the Toxilight assay (p-value = 9.74 × 10−9, 1.43 × 10−10 (h) and 7.79 × 10−10, 7.55 × 10−8 (i)). j, k, 293 T cells were co-transfected with SIGLEC12-FL-FLAG and TMPRSS4-HA for 24 h (j), and HT-29-SIGLEC12-KO-SIGLEC12-FLAG stable cell line was treated with TSE for 4 h with or without co-treatment with a protease inhibitor cocktail (PIC, cOmplete™, 1x final concentration) (k). Cell lysates and anti-FLAG immunoprecipitation samples were immunoblotted with the indicated antibodies. Data were plotted as the mean ± s.d., representative of n = 6 (b), n = 7 (i) or n = 9 (f and h); three independent experiments. Statistical analyses were performed using two-tailed t-test; ****p < 0.0001. Data are representative of three independent experiments (d, e, g, j and k). Panel a was created using Biorender (https://biorender.com).