Extended Data Fig. 9: SIGLEC12 is cleaved by TMPRSS4 to induce plasma membrane rupture during necroptosis.
From: SIGLEC12 mediates plasma membrane rupture during necroptotic cell death
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).
