Phospho-MLKL (Ser358) Antibody - #AF7420

Fig. 1: CD47 is overexpressed on necroptotic hepatocytes in the damaged liver of ALF models. A Biochemical evaluation (AST and ALT levels) of the ALF model after APAP induction (left). Representative images of H&E and CD47 staining of liver samples from an ALF model induced by APAP (right) (n = 4). B CD47 expression levels of cell populations within liver tissue (n = 4). C CD47 expression in hepatocytes from normal and APAP-ALF livers (n = 3). D Graphical representation of early apoptotic (Annexin V+/7-AAD−), late apoptotic/necroptotic (Annexin V+/7-AAD+), and necrotic (Annexin V−/7-AAD+) cell populations in liver hepatocytes from normal (n = 4) and APAP-ALF (n = 3) mice. E Expression of RIP3 and pMLKL in liver samples from normal and APAP-ALF groups. F Representative confocal images of liver sections from normal and APAP-ALF mice. Scale bar, 50 μm. G Scatter plot visualization of the Spearman correlation (R) between 121 necroptosis gene scoring and RNA levels of CD47 across samples. H STopover analysis to map the spatial overlap and interactions between cell types in liver tissue from normal and APAP-ALF mice. Highlights include 121 necroptosis gene scores (yellow) and macrophage RNA levels (blue), with overlapping areas shown in green. The plots below represent the three regions. I Violin plot visualizing SIRPα expression levels in different regions based on spatial correlation results. The numbers marked on the plot represent the median values of the SIRPα expression. Bar graph data are presented as mean ± SD. Statistical significance was determined by two-tailed unpaired Student’s t test (A), two-way ANOVA with Sidak’s post hoc test (B, D), and two-tailed test (G). Hepa hepatocytes, Immune immune cells, Endo endothelial cells. Source data are provided as a Source Data file.

Fig. 1: RS218 induced multiple death pathways in BMECs in vitro and in vivo. a Time-lapse microscopy of PI-stained (red) hBMECs infected with RS218 (MOI = 10), showing 8 min interval images taken at 60 × magnification. The 10 frames demonstrated the progression of cell death within 72 min before membrane rupture. White dashed lines indicate the boundaries of the cell membrane. Scale bar, 20 μm. b, c hBMECs stimulated with RS218 (MOI = 1 or 10) were used to determine cytotoxicity by LDH release (b, n = 3 biologically independent samples) and kinetics of cell death of PI penetration (c, n = 5 biologically independent samples). d, e hBMECs were pretreated with NSA or zVAD alone or in combination for 0.5 h, and cytotoxicity (d, n = 6 biologically independent samples) and cell death kinetics of PI penetration (e, n = 3 biologically independent samples) were assayed. f Flow cytometry of Annexin V-FITC and PI staining in hBMECs infected with RS218 (MOI = 1). Representative images of immunofluorescence represented morphological analysis of Annexin V-FITC and PI-labeled cells for the indicated times postinfection. Scale bar, 20 μm. g Immunoblot analysis of MLKL phosphorylation, GSDMD, caspase-3, and PARP1 cleavage in hBMECs infected with RS218 (MOI = 1) at the indicated times. Blots for GAPDH served as loading controls. +, indicated pharmacologically-induced death of hBMECs. FL, full-length. h Pro caspase-8 was co-immunoprecipitated with RIPK1, RIPK3, ZBP1 and ASC in uninfected or RS218-infected hBMECs (MOI = 1) for 3 h. i Immunofluorescence representative images of 2-day-old mice brain tissue sections 18 h after injection. The nucleus was labeled in blue, pMLKL or GSDMD-N or caspase-3 p17 in green, and endothelial cell marker CD31 in red. Scale bar, 20 μm. All experiments were representative of at least three independent experiments with similar results. Bars indicated the mean plus SD. Statistical significance was determined by one-way ANOVA with Tukey’s post-hoc test, with P-values denoted as follows: **** P 

Figure 3 Ir1-mediated PDT induces ferroptosis and necroptosis to initiate ICD in MDA-MB-231 cells. (a) Effects of pharmacological inhibitors on the antiproliferative efficacy of Ir1-PDT (n = 6). (b and c) Western blot analysis of protein expression profiles modulated by Ir1-PDT (n = 3). (d) Extracellular ATP levels in MDA-MB-231 cell supernatants (n = 3). (e and f) Immunofluorescence analysis of CRT (e) and HMGB1 (f) expression following Ir1-PDT treatment. Ir1: 0.1 µM, n = 3. Scale bar: 100 µm. Data are presented as mean ± SEM.

Fig. 4. PTX formulations inhibited macrophage PANoptosis. (A) Gene clustering analysis of PTX, FePTX, FePTX@CM-treated BMDMs. (B-C) GSEA analysis of transcriptomics results of BMDMs after LPS intervention. (D-E) Inflammatory-related pathway analysis in transcriptomics results of BMDMs after different PTX formulation interventions. (F) Heatmap of transcriptional profiling of inflammatory factors in BMDMs after PTX formulation interventions. (G) Results of qPCR sequencing (n = 4). (H) Cell viability of BMDMs with different treatments (n = 4). (I) PI staining images of BMDMs after LPS and FePTX@CM NPs treatment and its analysis (n = 4). Scale bar = 100 μm. (J) WB detection of the PANoptosis biomarkers in BMDMs upon treatment with FePTX@CM NPs. (K) TEM images of BMDMs after different treatments. Scale bar = 5 μm. (L) Co-IP analysis of PANoptosomes biomarkers in RAW264.7 after various interventions (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, N.s. = no significance. Data = mean ± SD.