Journal: Journal of Translational Autoimmunity

Article Title: Type I interferon-dependent and -independent signaling underlie autoantibody production in a murine lupus model

doi: 10.1016/j.jtauto.2026.100351

Figure Lengend Snippet: Induction of SLE autoantibodies occurs in the presence of LPS or Poly I:C, but not other TLR ligands. Wild type (WT) C57BL/6 mice received five immunizations with β2GPI in combination with one of the following TLR ligands: TLR2 : PAM2, FSL-1 (lipoprotein); TLR3 : Poly I:C (dsRNA); TLR4 : LPS; TLR7: IMQ (imiquimod); or TLR9 : CpG-ODN (ssDNA). Serum autoantibodies were detected by ELISA. Bars indicate the mean value +/standard error (S.E.) per group (n = 5 mice/group), and dots indicate values for individual mice. Autoantibody levels for control mice immunized with PBS and TLR ligand were OD 405 < 0.1. The OD 405 + 3 S.D. for unimmunized mice was ≤0.1 for all autoantibodies assayed. (A) Anti-β2GPI and anti-CL antibodies, and (B) hallmark SLE autoantibodies (anti-DNA, anti-Ro/SS-A, anti-La/SS-B, and anti-Sm) were analyzed statistically in separate groupings. For each of the two autoantibody categories, each different TLR ligand (immunized with β2GPI) was compared to the other TLR ligands by 2-way ANOVA, followed by a Tukey's multiple comparison test. ns = not significant. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Article Snippet: Lipopolysaccharide (LPS; Escherichia coli -derived, serotype O111:B4) was from List Biological Laboratories, Campbell, CA or Sigma-Aldrich Canada Co., Oakville, ON, and recombinant mouse IFN-β1 (carrier-free) was from Biolegend (San Diego, CA).

Techniques: Enzyme-linked Immunosorbent Assay, Control, Comparison

Journal: Journal of Translational Autoimmunity

Article Title: Type I interferon-dependent and -independent signaling underlie autoantibody production in a murine lupus model

doi: 10.1016/j.jtauto.2026.100351

Figure Lengend Snippet: Induction of SLE autoantibodies with LPS is dependent on TRIF and partially dependent on MyD88. WT, MyD88 −/− , or TRIF −/− mice were immunized with β2GPI and LPS; all mice received four immunizations. Serum autoantibodies were detected by ELISA. Bars indicate the mean value±S.E. per group (n = 5–6 mice/group/experiment), and dots indicate values for individual mice. Data are pooled from two separate experiments. The OD 405 + 3 S.D. for unimmunized mice was ≤0.1 for all autoantibodies assayed. (A) Anti-β2GPI and anti-CL antibodies, and (B) hallmark SLE autoantibodies (anti-DNA, anti-Ro/SS-A, anti-La/SS-B, and anti-Sm) were analyzed statistically in separate groupings. For each of the two autoantibody categories, mouse strains were compared by 2-way ANOVA, followed by a Dunnett's multiple comparison test. ∗∗∗∗P < 0.0001.

Article Snippet: Lipopolysaccharide (LPS; Escherichia coli -derived, serotype O111:B4) was from List Biological Laboratories, Campbell, CA or Sigma-Aldrich Canada Co., Oakville, ON, and recombinant mouse IFN-β1 (carrier-free) was from Biolegend (San Diego, CA).

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

Journal: Journal of Translational Autoimmunity

Article Title: Type I interferon-dependent and -independent signaling underlie autoantibody production in a murine lupus model

doi: 10.1016/j.jtauto.2026.100351

Figure Lengend Snippet: Induction of SLE autoantibodies is diminished in the absence of IRF3 and IFNaR. WT, IRF3 −/− , or IFNaR −/− mice were immunized with β2GPI and LPS; all mice received four immunizations. Serum autoantibodies were detected by ELISA. Bars indicate the mean value±S.E. per group (n = 5–6 mice/group/experiment), and dots indicate values for individual mice. Data are pooled from two separate experiments. The OD 405 + 3 S.D. for unimmunized mice was ≤0.1 for all autoantibodies assayed. (A) Anti-β2GPI and anti-CL antibodies, and (B) hallmark SLE autoantibodies (anti-DNA, anti-Ro/SS-A, anti-La/SS-B, anti-Sm, and anti-Sm/RNP) were analyzed statistically in separate groupings. For each of the two autoantibody categories, mouse strains were compared by 2-way ANOVA, followed by a Tukey's multiple comparison test. ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001.

Article Snippet: Lipopolysaccharide (LPS; Escherichia coli -derived, serotype O111:B4) was from List Biological Laboratories, Campbell, CA or Sigma-Aldrich Canada Co., Oakville, ON, and recombinant mouse IFN-β1 (carrier-free) was from Biolegend (San Diego, CA).

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

Journal: Journal of Translational Autoimmunity

Article Title: Type I interferon-dependent and -independent signaling underlie autoantibody production in a murine lupus model

doi: 10.1016/j.jtauto.2026.100351

Figure Lengend Snippet: Induction of hallmark SLE autoantibodies with LPS is enhanced in the presence of IFN-β in WT and TRIF-deficient, but not IRF3-deficient, mice. WT (A,B), TRIF −/− (C,D), and IRF3 −/− (E,F) mice were treated with IFN-β at the time of immunization with β2GPI and LPS and for two days after this immunization. All mice received five immunizations. Serum autoantibodies were detected by ELISA. Bars indicate the mean value±S.E. per group (n = 5 mice/group), and dots indicate values for individual mice. The OD 405 + 3 S.D. for unimmunized mice was ≤0.1 for all autoantibodies assayed. (A, C, E) Anti-β2GPI and anti-CL antibodies, and (B,D,F) hallmark SLE autoantibodies (anti-DNA, anti-Ro/SS-A, anti-La/SS-B, anti-Sm, and anti-Sm/RNP) were analyzed statistically in separate groupings. For each of the two autoantibody categories, mouse strains were compared by 2-way ANOVA, followed by a Tukey's multiple comparison test. ∗P < 0.05. ns = not significant.

Article Snippet: Lipopolysaccharide (LPS; Escherichia coli -derived, serotype O111:B4) was from List Biological Laboratories, Campbell, CA or Sigma-Aldrich Canada Co., Oakville, ON, and recombinant mouse IFN-β1 (carrier-free) was from Biolegend (San Diego, CA).

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

Journal: Journal of Translational Autoimmunity

Article Title: Type I interferon-dependent and -independent signaling underlie autoantibody production in a murine lupus model

doi: 10.1016/j.jtauto.2026.100351

Figure Lengend Snippet: Immunization with β2GPI + LPS induces cell cycle and interferon signaling pathways in dendritic cells (DCs). (A) Heatmap of normalized gene expression (transcripts per million [TPM]) of selected genes in DCs for all experimental samples. Genes included in the heatmap are the top 20 differentially expressed genes between β2GPI/LPS-immunized WT mice (n = 3) and each experimental group (WT [PBS-immunized, n = 3]; and β2GPI/LPS-immunized TRIF −/− [n = 4], IRF3 −/− [n = 5], and IFNaR −/− [n = 5] mice). (B) Volcano plot of differential gene expression by splenic DCs from β2GPI/LPS-immunized and PBS-immunized WT mice. (C–D) Bar plot (C) and normalized enrichment score plots (D) of selected enriched pathways (p < 0.05) determined by gene set enrichment analysis (GSEA) for genes differentially expressed by DCs from β2GPI/LPS-immunized versus PBS-immunized WT mice. GSEA was performed using the REACTOME database. All cell cycle-related pathways were combined and analyzed as a single composite enrichment score. (E) Immune response enrichment analysis (IREA) of genes upregulated in β2GPI + LPS-immunized, compared to PBS-immunized WT mice. Significantly enriched IFN-I-related cytokines are highlighted in yellow and IFN-II-related cytokines are highlighted in green. Statistical significance was determined by two-sided Wilcoxon rank-sum test, followed by the Benjamini-Hochberg correction for multiple comparisons. The top 10 enriched cytokine signatures are shown. The minimal threshold for significance is p < 0.05.

Article Snippet: Lipopolysaccharide (LPS; Escherichia coli -derived, serotype O111:B4) was from List Biological Laboratories, Campbell, CA or Sigma-Aldrich Canada Co., Oakville, ON, and recombinant mouse IFN-β1 (carrier-free) was from Biolegend (San Diego, CA).

Techniques: Protein-Protein interactions, Gene Expression

Journal: Journal of Translational Autoimmunity

Article Title: Type I interferon-dependent and -independent signaling underlie autoantibody production in a murine lupus model

doi: 10.1016/j.jtauto.2026.100351

Figure Lengend Snippet: Immunization with β2GPI + LPS induces cell cycle and interferon signalling pathways in macrophages. (A) Heatmap of normalized gene expression (transcripts per million [TPM]) of selected genes in macrophages for all experimental samples. Genes included in the heatmap are the top 20 differentially expressed genes between β2GPI/LPS-immunized WT mice (n = 3) and each experimental group (WT [PBS-immunized, n = 3]; and β2GPI/LPS-immunized TRIF −/− [n = 4], IRF3 −/− [n = 5], and IFNaR −/− [n = 5] mice). (B) Volcano plot of differential gene expression by splenic macrophages from β2GPI/LPS-immunized vs PBS-immunized WT mice. (C,D) Bar plot (C) and normalized enrichment score plots (D) of selected enriched pathways (p < 0.05) determined by gene set enrichment analysis (GSEA) for genes differentially expressed by macrophages from β2GPI/LPS-immunized vs PBS-immunized WT mice. GSEA was performed using the REACTOME database. All cell cycle-related pathways were combined and analyzed as a single composite enrichment score. (E) Immune response enrichment analysis (IREA) of genes upregulated in β2GPI + LPS-immunized, compared to PBS-immunized WT mice. Significantly enriched IFN-I-related cytokines are highlighted in yellow, IFN-II-related cytokines are highlighted in green, and NF-κB-dependent pro-inflammatory cytokines are highlighted in pink. Statistical significance was determined by two-sided Wilcoxon rank-sum test, followed by the Benjamini-Hochberg correction for multiple comparisons. The top 10 enriched cytokine signatures are shown. The minimal threshold for significance is p < 0.05.

Article Snippet: Lipopolysaccharide (LPS; Escherichia coli -derived, serotype O111:B4) was from List Biological Laboratories, Campbell, CA or Sigma-Aldrich Canada Co., Oakville, ON, and recombinant mouse IFN-β1 (carrier-free) was from Biolegend (San Diego, CA).

Techniques: Gene Expression

Journal: Journal of Translational Autoimmunity

Article Title: Type I interferon-dependent and -independent signaling underlie autoantibody production in a murine lupus model

doi: 10.1016/j.jtauto.2026.100351

Figure Lengend Snippet: TRIF- and IRF3-dependent genes upregulated in dendritic cells (DCs) following β2GPI/LPS-immunization are mostly IFN-I dependent. Genes upregulated in β2GPI/LPS-immunized WT (n = 3) vs PBS-immunized WT (n = 3) DCs were selected for downstream pathway analysis. (A) Venn diagram of the gene overlap between TRIF-dependent (upregulated in β2GPI/LPS-immunized WT vs TRIF −/− ; orange) and IRF3-dependent (upregulated in β2GPI/LPS-immunized WT vs IRF3 −/− ; green) genes. Genes that were both TRIF-and IRF3-dependent (blue circle) were selected for further analysis. (B,C) Pathway enrichment analysis of the 295 TRIF- and IRF3-dependent genes, using the PANTHER Gene Ontology: Biological Processes (B) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways databases (C). The top 10 differentially expressed pathways are shown for each analysis. (D) Immune responses enrichment analysis of the 295 TRIF- and IRF3-dependent genes. Significantly enriched IFN-I-related cytokines are highlighted in yellow and IFN-II-related cytokines are highlighted in green. Statistical significance was determined by two-sided Wilcoxon rank-sum test, followed by the Benjamini-Hochberg correction for multiple comparisons. The minimal threshold for significance is p < 0.05. (E) Volcano plot of differential gene expression for the 295 TRIF-and IRF3-dependent genes in β2GPI/LPS-immunized WT vs IFNaR −/− mice.

Article Snippet: Lipopolysaccharide (LPS; Escherichia coli -derived, serotype O111:B4) was from List Biological Laboratories, Campbell, CA or Sigma-Aldrich Canada Co., Oakville, ON, and recombinant mouse IFN-β1 (carrier-free) was from Biolegend (San Diego, CA).

Techniques: Gene Expression

Journal: Journal of Translational Autoimmunity

Article Title: Type I interferon-dependent and -independent signaling underlie autoantibody production in a murine lupus model

doi: 10.1016/j.jtauto.2026.100351

Figure Lengend Snippet: TRIF- and IRF3-dependent genes upregulated in macrophages following β2GPI/LPS-immunization are mostly IFN-I-dependent. Genes upregulated in β2GPI/LPS-immunized WT (n = 3) vs PBS-immunized WT (n = 3) macrophages were selected for downstream pathway analysis. (A) Venn diagram of the gene overlap between TRIF-dependent (upregulated in β2GPI/LPS-immunized WT vs TRIF −/− ; orange) and IRF3-dependent (upregulated in β2GPI/LPS-immunized WT vs IRF3 −/− ; green) genes. Genes that were both TRIF- and IRF3-dependent (blue circle) were selected for further analysis. (B,C) Pathway enrichment analysis of the 154 TRIF- and IRF3-dependent genes, using the PANTHER Gene Ontology: Biological Processes (B) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways databases (C). The top 10 differentially expressed pathways are shown for each analysis. (D) Immune responses enrichment analysis of the 154 TRIF- and IRF3-dependent genes. Significantly enriched IFN-I-related cytokines are highlighted in yellow and IFN-II-related cytokines and highlighted in green. Statistical significance was determined by two-sided Wilcoxon rank-sum test, followed by the Benjamini-Hochberg correction for multiple comparisons. The top 10 enriched cytokine signatures are shown. The minimal threshold for significance is p < 0.05. (E) Volcano plot of differential gene expression for the 154 TRIF-and IRF3-dependent genes in β2GPI/LPS-immunized WT vs IFNaR −/− mice.

Article Snippet: Lipopolysaccharide (LPS; Escherichia coli -derived, serotype O111:B4) was from List Biological Laboratories, Campbell, CA or Sigma-Aldrich Canada Co., Oakville, ON, and recombinant mouse IFN-β1 (carrier-free) was from Biolegend (San Diego, CA).

Techniques: Gene Expression

Journal: Journal of Translational Autoimmunity

Article Title: Type I interferon-dependent and -independent signaling underlie autoantibody production in a murine lupus model

doi: 10.1016/j.jtauto.2026.100351

Figure Lengend Snippet: TRIF- and IRF3-dependent genes with little to no IFNaR-dependence are associated with TNF production and signalling. (A) Venn diagram of the gene overlap between TRIF-dependent (upregulated in β2GPI/LPS-immunized WT vs TRIF −/− ; orange), IRF3-dependent (upregulated in β2GPI/LPS-immunized WT vs IRF3 −/− ; green), and IFNaR-dependent (upregulated in β2GPI/LPS-immunized WT vs IFNaR −/− ; yellow) genes in DCs. The 77 TRIF- and IRF3-dependent genes with little to no IFNaR-dependence (blue circle) were selected for further analysis. (B,C) Pathway enrichment analysis of the 77 TRIF- and IRF3-dependent genes with little to no IFNaR-dependency, using the PANTHER Gene Ontology: Biological Processes (B) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways databases (C). The top 10 differentially expressed pathways are shown for each analysis. (D) Immune response enrichment analysis of the of the 77 TRIF- and IRF3-dependent genes with little to no IFNaR-dependency. The significantly enriched NF-κB-dependent pro-inflammatory cytokine is highlighted in pink. Statistical significance was determined by two-sided Wilcoxon rank-sum test, followed by the Benjamini-Hochberg correction for multiple comparisons. The minimal threshold for significance is p < 0.05. (E) Venn diagram of the gene overlap between TRIF-dependent (upregulated in β2GPI/LPS-immunized WT vs TRIF −/− ; orange), IRF3-dependent (upregulated in β2GPI/LPS-immunized WT vs IRF3 −/− ; green), and IFNaR-dependent (upregulated in β2GPI/LPS-immunized WT vs IFNaR −/− ; yellow) genes in macrophages. The 6 TRIF- and IRF3-dependent genes with little to no IFNaR-dependence (blue circle) were selected for further analysis. (F) Transcript per million (TPM) of Tnf in macrophages for each experimental group. Data represent the mean ± SD. Statistical significance was determined by one-way ANOVA, followed by a Dunnett's post-hoc test. (G) C57BL/6 WT mice were injected intraperitoneally with β2GPI + PBS or β2GPI + LPS. TNF concentration was assayed in the serum by cytokine array assay (44-plex cytokine assay, Eve Technologies). Statistical significance was determined by Student's t-test. ns = not significant. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: Lipopolysaccharide (LPS; Escherichia coli -derived, serotype O111:B4) was from List Biological Laboratories, Campbell, CA or Sigma-Aldrich Canada Co., Oakville, ON, and recombinant mouse IFN-β1 (carrier-free) was from Biolegend (San Diego, CA).

Techniques: Injection, Concentration Assay, Cytokine Assay

Journal: Journal of Translational Autoimmunity

Article Title: Type I interferon-dependent and -independent signaling underlie autoantibody production in a murine lupus model

doi: 10.1016/j.jtauto.2026.100351

Figure Lengend Snippet: TRIF- and IRF3-dependent genes from both DCs and macrophages have an SLE-like signature. (A) Venn diagram of the overlap of upregulated TRIF-dependent (upregulated in β2GPI/LPS-immunized WT vs TRIF −/− ) and IRF3-dependent (upregulated in β2GPI/LPS-immunized WT vs IRF3 −/− ) genes between DCs (blue) and macrophages (red). The TRIF-and IRF3-dependent genes that were upregulated in both DCs and macrophages were selected for further analysis. (B,C) Pathway enrichment analysis of the 27 TRIF- and IRF3-dependent genes upregulated in both DCs and macrophages, using the PANTHER Gene Ontology: Biological Processes (B) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways databases (C). The top 10 differentially expressed pathways are shown for each analysis. (D) Immune responses enrichment analysis of the 27 TRIF- and IRF3-dependent genes upregulated in both DCs and macrophages. Significantly enriched IFN-I-related cytokines are highlighted in yellow and IFN-II-related cytokines are highlighted in green. Statistical significance was determined by two-sided Wilcoxon rank-sum test, followed by the Benjamini-Hochberg correction for multiple comparisons. The top 10 enriched cytokine signatures are shown. The minimal threshold for significance is p < 0.05. (E) Transcription factor enrichment analysis of the 27 TRIF- and IRF3-dependent genes upregulated in both DCs and macrophages. The top 10 transcription factors associated with this gene signature are included. Distance between transcription factors was adjusted for readability. (F) Disease signature enrichment analysis of the 27 TRIF- and IRF3-dependent genes upregulated in both DCs and macrophages. The top 10 enriched disease signatures from DiSignAtlas are included. The DiSignAtlas ID of the top 10 enriched disease signatures are as follows: DSA02284, DSA01156, DSA03951, DSA02253, DSA07100, DSA04468, DSA01254, DSA01302, DSA08191, and DSA03028.

Article Snippet: Lipopolysaccharide (LPS; Escherichia coli -derived, serotype O111:B4) was from List Biological Laboratories, Campbell, CA or Sigma-Aldrich Canada Co., Oakville, ON, and recombinant mouse IFN-β1 (carrier-free) was from Biolegend (San Diego, CA).

Techniques:

Journal: bioRxiv

Article Title: Hmgb1 release kinetics shape its extracellular functions during regulated cell death

doi: 10.64898/2026.05.15.725293

Figure Lengend Snippet: a Hmgb1–mCherry was expressed in various murine tissues. Tissue extracts were prepared from the indicated organs of 8-week-old wild-type or Hmgb1–mCherry Tg mice. The expression of Hmgb1–mCherry was analyzed by Western blotting with the indicated antibodies. The asterisk indicates the cross-reactive band. b Expression of Hmgb1–mCherry in peritoneal macrophages. Mice of the indicated genotypes were intraperitoneally injected with thioglycollate, and peritoneal cells were subsequently recovered by washing the peritoneal cavity with ice-cold PBS on Day 4 after injection. Isolated cells were stained with the indicated antibodies and analyzed by flow cytometry. The percentages of CD11b + F4/80 + cells (presumably peritoneal macrophages) are shown on the left. The percentages of mCherry-positive cells and their fluorescence intensities are shown as histograms on the right. c Peritoneal macrophages from Hmgb1–mCherry Tg mice were left untreated or primed with LPS (10 ng mL −1 ) for 3 h and then stimulated with nigericin (5 µM) for 1 h to induce pyroptosis or BV6 (1 μM) + zVAD (20 μM) to induce necroptosis in the absence or presence of the RIPK3 inhibitor GSK’872 (5 μM) for 4 h. The culture supernatants (Sups) or whole-cell lysates (WCLs) were analyzed by Western blotting with the indicated antibodies. P and M indicate the proform and mature form of IL-1β, respectively; F and N indicate the full-length and N-terminal fragment of Gsdmd, respectively. The asterisks indicate cross-reactive bands. All the results are representative of two or three independent experiments. Source data are provided as Supplementary Data 1.

Article Snippet: We purchased the following reagents: cisplatin (AG-CR1-3590, AdipoGen), BV6 (B4653, APExBIO), FVD506 (65-0866-14, Invitrogen), GSK’872 (530389, Merck), human HMGB1 (C-terminal His Tag, 10326-H08H, Sino Biological), LPS (434, List Labs), nigericin (AG-CN2-0020, AdipoGen), murine TNF (34-8321, eBioscience), and zVAD (3188-v, Peptide Institute).

Techniques: Expressing, Western Blot, Injection, Isolation, Staining, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Hmgb1 release kinetics shape its extracellular functions during regulated cell death

doi: 10.64898/2026.05.15.725293

Figure Lengend Snippet: Peritoneal macrophages isolated from Hmgb1–mCherry Tg mice were primed with LPS (10 ng mL −1 ) for 3 h and subsequently stimulated with nigericin (20 μM). The release of Hmgb1–mCherry and IL-1β was analyzed by LCI-S over 8 h. a Quantification of cells releasing both Hmgb1–mCherry and IL-1β (H and I), Hmgb1–mCherry alone (H alone), or IL-1β alone (I alone). Cells exhibiting each release pattern were counted, and the percentages were calculated relative to the total number of released cells. Results are mean ± SD from three independent experiments (H and I , n = 136 cells; H alone, 220 cells; I alone, 96 cells). b Relative Hmgb1–mCherry fluorescence intensities in H and I cells versus H alone cells. The fluorescence intensity of each individual cell was quantified by LCI-S. Each dot represents a single cell pooled from three independent experiments (H and I, n = 136 cells; H alone, n = 220 cells). Results are mean ± SD. a.u., arbitrary unit. c–f Representative image montages ( c, d ) and release kinetics ( e, f ) of short-duration ( c, e ) and long-duration ( d, f ) cells. The release of IL-1β (blue) and Hmgb1–mCherry (magenta), together with SYTOX uptake (green), was visualized by LCI-S. White arrows indicate the timing of SYTOX uptake and the onset of Hmgb1–mCherry and IL-1β release. Representative release kinetics of Hmgb1–mCherry (magenta) and IL-1β (blue) from short-duration ( e ) and long-duration cells ( f ) are shown. Time 0 indicates the addition of nigericin. g Dual modes of Hmgb1–mCherry release. The duration of Hmgb1–mCherry release was calculated as described in the Methods section and analyzed by k -means clustering, and two groups were identified: short-duration cells ( n = 41) and long-duration cells ( n = 26). h Single release mode of IL-1β. The release duration was calculated as in ( g ) and classified into a single cluster ( n = 91 cells). The results in g and h are representative of three independent experiments. i , Relative Hmgb1–mCherry fluorescence intensities in short-duration versus long-duration cells. Data are presented as mean ± SD (short-duration, n = 41; long-duration, n = 26). a.u., arbitrary unit. Statistical significance was assessed by the one-way ANOVA followed by Tukey’s multiple comparison test ( a ) or Mann–Whitney U test ( b, i ). Representative results from three independent experiments are shown ( c–h ). Source data are provided as Supplementary Data 1.

Article Snippet: We purchased the following reagents: cisplatin (AG-CR1-3590, AdipoGen), BV6 (B4653, APExBIO), FVD506 (65-0866-14, Invitrogen), GSK’872 (530389, Merck), human HMGB1 (C-terminal His Tag, 10326-H08H, Sino Biological), LPS (434, List Labs), nigericin (AG-CN2-0020, AdipoGen), murine TNF (34-8321, eBioscience), and zVAD (3188-v, Peptide Institute).

Techniques: Isolation, Fluorescence, Single Cell, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Hmgb1 release kinetics shape its extracellular functions during regulated cell death

doi: 10.64898/2026.05.15.725293

Figure Lengend Snippet: Peritoneal macrophages isolated from Hmgb1–mCherry Tg mice were primed with LPS (10 ng mL −1 ) for 3 h and subsequently stimulated with BV6 (1 μM) + zVAD (20 μM). The release of Hmgb1–mCherry and IL-1β was monitored by LCI-S over 8 h. a Quantification of cells releasing both Hmgb1–mCherry and IL-1β (H and I), Hmgb1–mCherry alone (H alone), or IL-1β alone (I alone), based on the release patterns as shown in . Results are mean ± SD from three independent experiments (H and I, n = 134 cells; H alone , n = 178 cells; I alone , n = 67 cells). b Relative Hmgb1–mCherry fluorescence intensities quantified by LCI-S. Each dot represents a single cell pooled from three independent experiments (H and I, n = 134 cells; H alone , n = 178 cells). Data are shown as mean ± SD. a.u., arbitrary unit. c–f Representative image montages ( c, d ) and release kinetics ( e, f ) of short-duration and long-duration cells. The release of Hmgb1–mCherry (magenta) and IL-1β (blue), together with SYTOX uptake (green), was visualized by LCI-S. White arrows indicate the timing of SYTOX positivity and the onset of IL-1β and Hmgb1–mCherry release. Representative release kinetics of Hmgb1–mCherry (magenta) and IL-1β (blue) from short-duration ( e ) and long-duration cells ( f ) are shown. Time 0 indicates the addition of nigericin. g Dual modes of Hmgb1–mCherry release. The duration of Hmgb1–mCherry release was calculated as described in the Methods section and analyzed by k -means clustering, and two groups were identified: short-duration cells ( n = 42) and long-duration cells ( n = 16). h Single release mode of IL-1β. The release duration was calculated as in ( g ) and classified into a single cluster ( n = 77 cells). Representative data from three independent experiments are shown. i Relative Hmgb1–mCherry fluorescence intensities in short-duration versus long-duration cells. Data are presented as mean ± SD (short-duration, n = 42; long-duration, n = 16). a.u., arbitrary unit. Statistical significance was assessed by the one-way ANOVA with Tukey’s multiple comparison test ( a ), Mann–Whitney U test ( b ), or two-tailed unpaired Student’s t -test ( i ). Representative results from three independent experiments are shown ( c-h ). Source data are provided as Supplementary Data 1.

Article Snippet: We purchased the following reagents: cisplatin (AG-CR1-3590, AdipoGen), BV6 (B4653, APExBIO), FVD506 (65-0866-14, Invitrogen), GSK’872 (530389, Merck), human HMGB1 (C-terminal His Tag, 10326-H08H, Sino Biological), LPS (434, List Labs), nigericin (AG-CN2-0020, AdipoGen), murine TNF (34-8321, eBioscience), and zVAD (3188-v, Peptide Institute).

Techniques: Isolation, Fluorescence, Single Cell, Comparison, MANN-WHITNEY, Two Tailed Test