Journal: Journal of Extracellular Vesicles

Article Title: C1qc Mediates Blood–Brain Barrier Disruption in a Mouse Model of Intracerebral Haemorrhage Through Microglia‐Derived Migrasomes

doi: 10.1002/jev2.70377

Figure Lengend Snippet: C1qc mediates the effects of migrasomes on the BBB via the Nlrp3–Caspase 1–GSDMD pathway. (A) Immunoblots revealed C1qc, Nlrp3, cleaved caspase‐1, and GSDMD‐N protein expression levels in C1qc flox/flox model mice injected with AAV–Iba1–Cre or AAV–con ( n = 5). Densitometric quantification of protein levels; the data are presented as the mean SD; ** p < 0.01, *** p < 0.001, **** p < 0.0001. (B) Immunofluorescence staining for GSDMD–N and the endothelial cell marker CD31 in the perihaematomal area of C1qc flox/flox model mice injected with AAV–Iba1–Cre or AAV–con ( n = 5). Normalized quantitative measurement of GSDMD‐N fluorescence intensity relative to CD31 area. The data are presented as the mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (C) Immunofluorescence staining for cleaved caspase‐1 and the endothelial cell marker CD31 in the perihaematomal area of C1qc flox/flox model mice injected with AAV–Iba1–Cre or AAV–con ( n = 5). Fluorescence signal density from cleaved caspase‐1, normalized against the CD31 area, was analysed quantitatively. The data are expressed as ± SD; *** p < 0.001, **** p < 0.0001. (D) GSDMD‐N staining of endothelial cells that were treated as described in Figure ( n = 3). Quantification of the fluorescence intensity of GSDMD‐N of endothelial cells in the vehicle + microglia‐secreting migrasome group, the hemin + microglia‐secreting migrasome group, and the hemin + siC1qc microglia‐secreting migrasome group were also quantified. Data are presented as the mean ± SD with the indicated significance, * p < 0.05, ** p < 0.01, *** p < 0.001. (E) Cleaved caspase‐1 staining of endothelial cells that were treated as described previously in Figure ( n = 3). Quantification of the fluorescence intensity of Cleaved caspase‐1 of endothelial cells in the vehicle + microglial migrasome groups, hemin + microglial migrasome group, and hemin + siC1qc microglial migrasome group were also quantified. The data are presented as the means ± SDs; * p < 0.05, ** p < 0.01. (F) Immunoblots showing the levels of the Nlrp3 and GAPDH proteins in endothelial cells that were treated as described in Figure ( n = 6). Densitometric quantification of protein levels. The data are presented as the mean ± SD; *** p < 0.001. (G) Claudin‐5 staining of endothelial cells that were treated with MCC950 and microglial migrasomes without C1qc ( n = 5). Quantification of the fluorescence intensity of Claudin‐5. The data are presented as the mean ± SD; ns, no significant difference; ** p < 0.01, *** p < 0.001. (H) GSDMD‐N immunofluorescence in endothelial cells treated with migrasomes, C1qc‐deficient migrasomes, or C1qc‐deficient migrasomes plus Nigericin. Quantification of the fluorescence intensity of GSDMD‐N. The data are presented as the mean ± SD; ** p < 0.01, *** p < 0.001. (I) Claudin‐5 immunofluorescence in endothelial cells treated with migrasomes, C1qc‐deficient migrasomes, or C1qc‐deficient migrasomes plus Nigericin. Quantification of the fluorescence intensity of Claudin‐5. The data are presented as the mean ± SD; * p < 0.05, *** p < 0.001. (J) Immunoblots showing the levels of the GSDMD‐N, Claudin‐5 and GAPDH proteins in endothelial cells that were treated with migrasomes at different time points ( n = 6). Densitometric quantification of protein levels. The data are presented as the mean ± SD; ns = no significant difference, ** p < 0.01, *** p < 0.001. (K) Cell viability dramatically decreased endothelial cells that were treated with migrasomes at different time points ( n = 3). Data are shown as the means ± SD with significance levels; ns = no significant difference, * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: Ten milligrams of the inflammasome inhibitor MCC950 (Nlrp3 inhibitor)(Selleck Chemicals, Houston, TX, USA) was dissolved in 1 mL of 1× phosphate‐buffered saline (PBS).

Techniques: Western Blot, Expressing, Injection, Immunofluorescence, Staining, Marker, Fluorescence

Journal: iScience

Article Title: Linear ubiquitin chain assembly complex contributes to NLRP3-mediated pyroptotic cell death

doi: 10.1016/j.isci.2026.116448

Figure Lengend Snippet: Pyroptosis execution drives the formation of linear ubiquitin chains (A) Workflow used to induce pyroptosis in THP-1 monocytic cells. (B) Immunoblots of indicated proteins in lysates and supernatants of THP-1 cells primed with 1 μg/mL of LPS for 3 h before treatment with 10 μM of nigericin as indicated. Where indicated, cells were pre-treated with 1 μM of MCC-950 for 30 min before nigericin addition. Arrowheads indicate proteins and protein-cleaved fragments. The positions of molecular weights ( M r ) are indicated. (C) Immunoblots of THP-1 treated and analyzed as in (B). (D) Immunoblots of indicated proteins in lysates of A549 cells treated with 10 μM of nigericin. Lysates from untreated THP-1 cells served as a control. Quantification for Met-1 ubiquitination levels over loading (GAPDH). ( n = 3; mean ± SEM; ∗∗ p < 0.01; ANOVA). (E) THP-1 cells stably expressing HMGB1-Lucia deleted with sgRNAs for GSDMD (GSDMD sg ) or their control counterparts (Ctrl sg ) were treated and analyzed as in (B). Sup., supernatants. (F) The release of HMGB1-Lucia was assessed in the supernatants of cells as in (E). ( n = 3; mean ± SEM; ∗∗∗∗ p < 0.0001; t test). All the presented data are representative of at least three independent experiments.

Article Snippet: MCC-950 , Invivogen , Cat#inh-mcc.

Techniques: Ubiquitin Proteomics, Western Blot, Control, Stable Transfection, Expressing, Luciferase

Journal: iScience

Article Title: Linear ubiquitin chain assembly complex contributes to NLRP3-mediated pyroptotic cell death

doi: 10.1016/j.isci.2026.116448

Figure Lengend Snippet: LUBAC and linear ubiquitination drive pyroptosis (A) Immunoblots of indicated proteins in lysates and supernatants (Sup.) of THP-1 cells deleted with sgRNAs for HOIP (HOIP sg ) or their control counterparts (Ctrl sg ) primed with 1 μg/mL of LPS for 3 h before treatment with 10 μM of nigericin as indicated. Arrowheads indicate proteins and protein-cleaved fragments. The positions of molecular weights ( M r ) are indicated. (B) THP-1 deleted with sgRNAs for HOIL-1 (HOIL-1 sg ) or their control counterparts (Ctrl sg ) were treated and analyzed as in (A). (C) qPCR analysis of HOIP and HOIL-1 in the indicated THP-1 cell genotypes ( n = 3; mean ± SEM; ∗ p < 0.05; t test). (D) qPCR analysis of pro-IL-1β in the indicated THP-1 cell genotypes primed with 1 μg/mL of LPS for 3 h ( n = 3; mean ± SEM; ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ANOVA). (E) Immunoblots of indicated proteins lysates and supernatants (Sup.) of THP-1 cells treated as in (A). Arrowheads indicate proteins and protein-cleaved fragments. The positions of molecular weights ( M r ) are indicated. (F) PMA-differentiated THP-1 Ctrl sg , HOIP sg , and HOIL-1 sg were stimulated with 10 μM of nigericin in the presence of 100 nM of SYTOX Green. Incucyte acquisition was performed every 30 min to assess green object count ( n = 3; mean ± SEM; ∗∗ p < 0.01, ∗∗∗ p < 0.001 at end time point; ANOVA). (G) THP-1 were primed with LPS for 3 h, pre-treated with 30 μM of HOIPIN-1 or HOIPIN-8 for 30 min, then activated with 10 μM of nigericin as indicated and analyzed as in (A). Arrowheads indicate proteins and protein-cleaved fragments. The positions of molecular weights ( M r ) are indicated. (H) iBMDM cells were treated as in (G). (I) THP-1 cells stably expressing HMGB1-Lucia were treated as in (G). The release of HMGB1-Lucia was assessed in cell supernatants. ( n = 3; mean ± SEM; ∗∗∗∗ p < 0.0001; ANOVA). (J) PMA-differentiated THP-1 were pre-treated with 30 μM of HOIPIN-1, HOIPIN-8, or 1 μM of MCC-950, then treated with 10 μM of nigericin in the presence of 100 nM of SYTOX Green. Incucyte acquisition was performed as in (F) ( n = 3; mean ± SEM; ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001 at end time point; ANOVA). The presented data are representative of at least three independent experiments.

Article Snippet: MCC-950 , Invivogen , Cat#inh-mcc.

Techniques: Ubiquitin Proteomics, Western Blot, Control, Analysis, Stable Transfection, Expressing, RNA Expression

Journal: iScience

Article Title: Linear ubiquitin chain assembly complex contributes to NLRP3-mediated pyroptotic cell death

doi: 10.1016/j.isci.2026.116448

Figure Lengend Snippet:

Article Snippet: MCC-950 , Invivogen , Cat#inh-mcc.

Techniques: Cell-Signaling, Ubiquitin Proteomics, Recombinant, Electroporation, Protease Inhibitor, Western Blot, Purification, cDNA Synthesis, SYBR Green Assay, Luciferase, LDH Cytotoxicity Assay, Software, Imaging, Multiwell Plates, Optogenetics

Journal: EMBO Reports

Article Title: Caspase-3 cleaves CYLD to restrict interferon signaling during mitochondrial apoptosis

doi: 10.1038/s44319-026-00876-4

Figure Lengend Snippet: Related to Fig. . ( A ) Workflow to induce pyroptosis in monocytic cells. ( B ) THP-1 cells stably expressing HMGB1-Lucia deleted with sgRNAs for CYLD (CYLD sg ), CASP1 (CASP1 sg ), GSDMD (GSDMD sg ), or their control counterparts (Ctrl sg ) were primed with LPS (1 μg/mL) for 3 h before stimulation with Nigericin (10 μM) for 1 h. The release of HMGB1-Lucia was assessed in cell supernatants ( n = 3; mean ± SEM; **** P < 0.0001; two-way ANOVA). P values for each comparison: Ctrl sg vs CYLD sg : P < 0.0001; Ctrl sg vs CASP1 sg : P < 0.0001; Ctrl sg vs GSDMD sg : P < 0.0001. ( C ) Immunoblot analysis of THP-1 cells primed or not with LPS (1 μg/mL) for 3 h before treatment with Nigericin (10 μM) or Raptinal (10 μM) for 1 h. When indicated, cells were pretreated with the NLRP3 inhibitor MCC950 (2 μM) for 30 min. Closed and open symbols indicate full-length and cleaved CYLD. ( D ) Immunoblot analysis of Ctrl sg and CASP1 sg THP-1 cells treated as in ( B ). * indicates a nonspecific band. The presented immunoblots are representative of at least three independent experiments. .

Article Snippet: LPS (tlrl-peklps), Nigericin (tlrl-nig), and MCC950 (inh-mcc) were obtained from Invivogen.

Techniques: Stable Transfection, Expressing, Control, Comparison, Western Blot

Journal: EMBO Reports

Article Title: Caspase-3 cleaves CYLD to restrict interferon signaling during mitochondrial apoptosis

doi: 10.1038/s44319-026-00876-4

Figure Lengend Snippet: Reagents and tools table

Article Snippet: LPS (tlrl-peklps), Nigericin (tlrl-nig), and MCC950 (inh-mcc) were obtained from Invivogen.

Techniques: Ubiquitin Proteomics, Sequencing, Recombinant, Transfection, Software