Journal: iScience

Article Title: Antigen-directed single domain antibody-based TNFR1 agonists elicit preferential killing of HER2-overexpressing cancer cells

doi: 10.1016/j.isci.2026.115327

Figure Lengend Snippet: HER2-targeted TNFR1-agonists induce TNF-like caspase-1, caspase-3, and caspase-8 activation decoupled from NF-κB signaling in MCF-7 cells (A–C) Caspase-1/3/8 activation of HER2-expressing MCF-7 cells by ICM11 derivatives compared to (rh)TNF. MCF-7 cells were stimulated with increasing compound concentrations for 72 h. Caspase activities were detected intracellularly with FAM-FLICA(R) Caspase 1 Assay Kit (Biomol), CaspaTag Caspase-3 In situ Assay Kit (Merck Millipore), and CaspaTag Caspase-8 In Situ Assay Kit (Merck Millipore). Caspase-1/3/8 activation was normalized to (rh) TNF. (D) NF-κB activation in MCF-7 cells triggered by ICM11 derivatives and (rh) TNF. MCF-7 cells were stimulated with increasing concentrations of ICMs and (rh) TNF for 40 min. NF-κB was stained intracellularly with AF488-labeled anti-NF-κB staining antibody (BD) after lysis, fixation, and permeabilization of cells. (E) Remaining relative cell death of MCF-7 cells after treatment with ICM11 derivatives or (rh)TNF in the presence or absence of caspase inhibitors. MCF-7 cells were incubated with caspase-1 inhibitor (InvivoGen), caspase-3 inhibitor (R&D Systems), caspase-8 inhibitor (InvivoGen), or pan-caspase inhibitor (InvivoGen) at 50 μM and a fixed (rh) TNF or ICM concentration of 5 nM for 72 h. Killing was monitored by green fluorescence signal with SYTOX Green Dead Cell Stain and normalized to (rh) TNF signal. Mean values ±SEM of four independent experiments for each figure are shown. ∗∗∗∗ p < 0.0001,∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 calculated by utilizing two-way ANOVA multiple analyses and Bonferroni test.

Article Snippet: pan-caspase inhibitor (zVAD-FMK) , InvivoGen , Cat#tlrl-vad.

Techniques: Activation Assay, Expressing, In Situ, Staining, Labeling, Lysis, Incubation, Concentration Assay, 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: 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 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