Journal: Fluids and Barriers of the CNS

Article Title: Notch signaling activation reduces vesicular endocytosis in human pluripotent stem cell-derived CNS-like endothelial cells

doi: 10.1186/s12987-025-00754-6

Figure Lengend Snippet: Immunostaining of unedited hPSC-EPCs and hPSC-CECs with lentiviral constitutive N1ICD overexpression: Immunocytochemistry (ICC) analysis of various markers in hPSC-EPCs treated with DMSO, CHIR (hPSC-CECs), CHIR and GFP LV, or CHIR and N1ICD LV on D11 after 6 days of culture in hECSR supplemented with DMSO or CHIR and 4 days following transduction with either GFP or N1ICD LV. (A) Representative images of ICC analysis for caveolin-1, CD31, and GFP in various treatment conditions. Only LV transduced cells expressed GFP. Hoechst nuclear counterstain is overlaid in all images. Scale bar: 200 μm. (B) Quantification of caveolin-1 mean gray value (MGV) in different conditions from (A), normalized to Hoechst MGV. (C) Representative images of ICC analysis for PLVAP and CD31 in indicated treatment conditions. GFP staining not shown. Hoechst nuclear counterstain overlaid in all images. Scale bar: 200 μm. (D) Quantification of PLVAP MGV in indicated conditions from (C), normalized to Hoechst MGV. (E) Representative images of ICC analysis for GLUT-1 and CD31 in indicated treatment conditions. Hoechst nuclear counterstain overlaid in all images. Scale bar: 200 μm. (F) Quantification of GLUT-1 MGV in indicated conditions from (C), normalized to Hoechst MGV. In (B) , (D) , and (F) , points represent n = 3 biological replicates from one differentiation of IMR90-4 iPSC-derived EPCs. Horizontal bars indicate mean. Hoechst-normalized relative fluorescence for each of the three markers was further normalized within each analysis such that the mean of the DMSO condition was equal to 1. Statistical analyses were performed on Hoechst-normalized data; P-values: One-way ANOVA followed by Tukey’s HSD test

Article Snippet: Lentivirus encoding N1ICD -IRES- GFP (denoted N1ICD LV) and IRES- GFP negative control (denoted GFP LV) constructs were produced in HEK293TN cells (System Biosciences, Palo Alto, CA).

Techniques: Immunostaining, Over Expression, Immunocytochemistry, Transduction, Staining, Derivative Assay, Fluorescence

Journal: Fluids and Barriers of the CNS

Article Title: Notch signaling activation reduces vesicular endocytosis in human pluripotent stem cell-derived CNS-like endothelial cells

doi: 10.1186/s12987-025-00754-6

Figure Lengend Snippet: Differentiation and validation of PB-TRE-N1ICD hPSC-CECs with Dox-inducible N1ICD overexpression: (A) Protocol for differentiation of hPSCs to EPCs, followed by MACS sorting, hygromycin reselection, CHIR treatment to derive CECs and doxycycline treatment to overexpress N1ICD . (B) Schematic of the doxycycline inducible N1ICD overexpressing transposon construct. The N1ICD cassette follows a TRE3G doxycycline inducible promoter. A cassette encoding the Tet-On 3G protein follows an EF-1α core promoter, resulting in constitutive expression of this gene. Tet-On 3G must associate with doxycycline to bind to the TRE3G inducible promoter. Also following the EF-1α promoter and Tet-On 3G are self-cleaving 2A peptide linker (T2A) followed by a hygromycin resistance gene (HygR). The top portion of the schematic indicates that during differentiation of hPSCs to CECs, the TRE3G promoter is not active due to the absence of doxycycline. In the bottom portion, hPSC-CECs that have been differentiated and sorted can be treated with doxycycline to activate N1ICD overexpression. (C) Representative flow cytometry plots of D5 hPSC-EPCs derived from a population of N1ICD overexpressing IMR90-4 iPSCs with heterogeneous copy numbers of the integrated construct shown in (B). Graphs show percentage of CD31+/CD34 + EPCs before (pre-MACS) and after (post-MACS) sorting. (D) Western blotting analysis of D15 hPSC-derived PB-TRE-N1ICD 10(+ CR2) clonal EPCs treated with CHIR (hPSC-CECs), with or without doxycycline. Membranes blotted for Notch1 full length (N1 FL) protein and intracellular domain (N1ICD), as well as β-actin. Predicted approximate molecular weights of each detected protein are indicated on the right-hand side. (E) N1 FL and (F) N1ICD, normalized to respective input control (β-actin) band intensities. In all Western blot analyses, points represent n = 4 biological replicates from one differentiation of IMR90-4 PB-TRE-N1ICD 10(+ CR2) iPSC-derived CECs. Bars indicate mean ± SD. β-actin-normalized band intensities were further normalized within each analysis such that the mean of the CHIR + PBS condition was equal to 1. Statistical analyses were performed on β-actin-normalized data; P-values: Student’s t -test

Article Snippet: Lentivirus encoding N1ICD -IRES- GFP (denoted N1ICD LV) and IRES- GFP negative control (denoted GFP LV) constructs were produced in HEK293TN cells (System Biosciences, Palo Alto, CA).

Techniques: Biomarker Discovery, Over Expression, Construct, Expressing, Flow Cytometry, Derivative Assay, Western Blot, Control

Journal: Fluids and Barriers of the CNS

Article Title: Notch signaling activation reduces vesicular endocytosis in human pluripotent stem cell-derived CNS-like endothelial cells

doi: 10.1186/s12987-025-00754-6

Figure Lengend Snippet: Immunostaining of PB-TRE-N1ICD hPSC-CECs: Immunocytochemistry (ICC) analysis of indicated markers in PB-TRE-N1ICD hPSC-CECs on D15 after 10 days of culture in hECSR supplemented with CHIR and 1:1000 diluted DPBS from D5 to D11 and 4 days of CHIR and doxycycline (1 µg/mL) or PBS treatment from D11 to D15. (A) Representative images of ICC analysis for caveolin-1 and CD31. Hoechst nuclear counterstain is overlaid in all images. Scale bar: 200 μm. (B) Quantification of caveolin-1 mean gray value (MGV) in different conditions from ( A ), normalized to Hoechst MGV. (C) Representative images of ICC analysis for PLVAP and CD31. Hoechst nuclear counterstain is overlaid in all images. Scale bar: 200 μm. (D) Quantification of PLVAP MGV in different conditions from (C), normalized to Hoechst MGV. (E) Representative images of ICC analysis for GLUT-1 and CD31. Hoechst nuclear counterstain is overlaid in all images. Scale bar: 200 μm. (F) Quantification of GLUT-1 MGV in different conditions from ( C ), normalized to Hoechst MGV. In ( B ), ( D ), and ( F ), points represent n = 4 biological replicates from one differentiation of IMR90-4 PB-TRE-N1ICD 10(+ CR2) hPSC-CECs. Horizontal bars indicate mean ± SD. Hoechst-normalized relative fluorescence for each of the three markers was further normalized within each analysis such that the mean of the PBS condition was equal to 1. Statistical analyses were performed on Hoechst-normalized data; P-values: Student’s t test

Article Snippet: Lentivirus encoding N1ICD -IRES- GFP (denoted N1ICD LV) and IRES- GFP negative control (denoted GFP LV) constructs were produced in HEK293TN cells (System Biosciences, Palo Alto, CA).

Techniques: Immunostaining, Immunocytochemistry, Fluorescence

Journal: Fluids and Barriers of the CNS

Article Title: Notch signaling activation reduces vesicular endocytosis in human pluripotent stem cell-derived CNS-like endothelial cells

doi: 10.1186/s12987-025-00754-6

Figure Lengend Snippet: FACS sorting and bulk RNA-sequencing of hPSC-CECs with doxycycline-inducible N1ICD overexpression: (A) Schematic of culture timeline and FACS sorting for PB-TRE-N1ICD 10(+ CR2) hPSC-CECs treated with or without doxycycline to overexpress N1ICD . Cells in each PBS- ( n = 4) and Dox-treated ( n = 3) sample were sorted by FACS into subpopulations based on CD144 (VE-cadherin) expression. Lower boundary for CD144 high gate was set based on majority of PBS-treated hPSC-CECs and unedited hPSC-CECs cultured in the presence of CHIR alone. Lower boundary for CD144 low gate was set based on CD144 antibody-stained undifferentiated hPSCs and isotype control antibody-stained edited hPSC-CECs. Gated plots show representative distributions of PBS- and Dox-treated edited hPSC-CECs into CD144 high and CD144 low expressing subpopulations. (B) PCA plot showing relative differences in transcriptomic profiles of bulk RNA-sequenced subpopulations from (A), including PBS-treated CD144 high (PBS_CD144hi), Dox-treated CD144 high (Dox_CD144hi), and Dox-treated CD144 low (Dox_CD144lo). Raw bulk RNA-sequencing data were pre-processed using the Galaxy pipeline (usegalaxy.org) discussed in Materials & Methods and analyzed in RStudio using the DESeq2. (C) Normalized expression (transcripts per million, TPM) of NOTCH1 and HEYL from each bulk RNA-sequenced subpopulation discussed in ( A ) and ( B ). Statistics calculated by one-way ANOVA with post-hoc Tukey’s test, significant comparisons highlighted in red. (D) Heatmap of row-normalized TPM expression for Notch signaling-related genes that are significantly differentially expressed (FDR < 0.05) between the 3 subpopulations. Selected genes obtained from KEGG and Hallmark Notch signaling gene sets via MSigDB ( https://www.gsea-msigdb.org/gsea/msigdb ). Row z-scores were calculated for each gene by subtracting mean expression across all samples from normalized expression for a specific sample and dividing the result by the standard deviation. Row z-scores range from bright red, indicating high expression, and dark blue, indicating low expression. Heatmap was produced in RStudio using the pheatmap package

Article Snippet: Lentivirus encoding N1ICD -IRES- GFP (denoted N1ICD LV) and IRES- GFP negative control (denoted GFP LV) constructs were produced in HEK293TN cells (System Biosciences, Palo Alto, CA).

Techniques: RNA Sequencing, Over Expression, Expressing, Cell Culture, Staining, Control, Standard Deviation, Produced

Journal: Fluids and Barriers of the CNS

Article Title: Notch signaling activation reduces vesicular endocytosis in human pluripotent stem cell-derived CNS-like endothelial cells

doi: 10.1186/s12987-025-00754-6

Figure Lengend Snippet: Transcriptomic comparison of PB-TRE-N1ICD hPSC-CECs ± Dox to hPSC-derived, primary or immortalized endothelial and non-endothelial cell types and to in vivo BMECs: (A) Analysis of bulk transcriptomic data of subpopulations of PB-TRE-N1ICD 10(+ CR2) hPSC-CEC subpopulations using PACNet classification scores to identify similarity to generic cell types in a training data set. Sample replicates from left to right: PBS-treated, CD144-high (PBS_CD144hi); doxycycline-treated, CD144-high (Dox_CD144hi); doxycycline-treated, CD144-low (Dox_CD144lo); random cell type (rand) auto-generated by PACNet software. Classification score colors ranging from black to yellow indicate no transcriptomic resemblance to 100% transcriptomic match, respectively, to training data cell types indicated on y-axis. (B) PACNet analysis of primary human brain microvascular endothelial cells (HBMEC), immortalized hCMEC.D3 brain endothelial cells (hCMEC_D3), and primary human aortic endothelial cells (Haortic) relative to training set cell types. All analyses in (A) and (B) were performed using the PACNet webtool ( https://cahanlab.org/resources/agnosticCellNet_web/ ). (C) Heatmap of transcript abundance (log 2 [TPM + 1]) of indicated endothelial, mesenchymal, fibroblast, and epithelial genes across samples with different degrees of N1ICD overexpression. Heatmap columns indicate sample/replicate and rows indicate genes. Cell color indicates transcript abundance, ranging from blue (lowest expression) to red (highest). Heatmaps were generated using the pheatmap package in R. (D) PCA plot combining several in vivo and in vitro transcriptomic datasets, including: pseudo-bulked in vivo adult brain capillary endothelial cell scRNA-seq data (Yang et al., 2022) ; pseudo-bulked in vivo embryonic brain capillary endothelial cells scRNA-seq data (Crouch et al., 2022) ; in vitro primary/immortalized brain endothelial and primary peripheral endothelial cells (similar to those presented in [B]) bulk RNA-seq data (Qian et al., 2017) ; in vitro Passage 1 hPSC-derived endothelial progenitor cells (EPCs) treated with CHIR and hPSC-derived smooth muscle-like cells (SMLCs) bulk RNA-seq data (Gastfriend et al., 2021) ; in vitro primary cells representing different developmental lineages bulk RNA-seq data (Pandey, 2022, GSE190615 ); in vitro PB-TRE-N1ICD hPSC-CECs with or without doxycycline bulk RNA-seq data (this study). PCA plot was generated using the DESeq2 package in R

Article Snippet: Lentivirus encoding N1ICD -IRES- GFP (denoted N1ICD LV) and IRES- GFP negative control (denoted GFP LV) constructs were produced in HEK293TN cells (System Biosciences, Palo Alto, CA).

Techniques: Comparison, Derivative Assay, In Vivo, Generated, Software, Over Expression, Expressing, In Vitro, RNA Sequencing

Journal: Fluids and Barriers of the CNS

Article Title: Notch signaling activation reduces vesicular endocytosis in human pluripotent stem cell-derived CNS-like endothelial cells

doi: 10.1186/s12987-025-00754-6

Figure Lengend Snippet: Normalized expression of selected BBB and cell identity-related genes from bulk RNA-seq of edited PB-TRE-N1ICD 10(+ CR2) hPSC-CECs ± Dox subpopulations: (A-M) Comparison of normalized gene expression (TPM) between bulk RNA-sequenced PBS- and Dox-treated subpopulations for indicated genes. In all analyses, points represent n = 3 or n = 4 biological replicates depending on treatment condition from one differentiation of IMR90-4 PB-TRE-N1ICD 10(+ CR2) hPSC-CECs. Bars indicate mean ± SD. Relative gene expression values are shown in TPM, and statistical analyses were performed on TPM data; P-values < 0.05 by one-way ANOVA with post-hoc Tukey’s test are highlighted in red

Article Snippet: Lentivirus encoding N1ICD -IRES- GFP (denoted N1ICD LV) and IRES- GFP negative control (denoted GFP LV) constructs were produced in HEK293TN cells (System Biosciences, Palo Alto, CA).

Techniques: Expressing, RNA Sequencing, Comparison, Gene Expression

Journal: Fluids and Barriers of the CNS

Article Title: Notch signaling activation reduces vesicular endocytosis in human pluripotent stem cell-derived CNS-like endothelial cells

doi: 10.1186/s12987-025-00754-6

Figure Lengend Snippet: N1ICD overexpression reduces fluorescent albumin accumulation by inhibiting caveolae-mediated endocytosis: (A) Representative live cell epifluorescence microscopy images of 10(+ CR2) PB-TRE-N1ICD hPSC-CECs cultured in hECSR + CHIR, with PBS or Dox, and PBS-cultured cells with methyl-β-cyclodextrin (MβCD, inhibitor of caveolae-mediated endocytosis) pretreatment that were incubated at 37 °C with bovine serum albumin, or BSA, conjugated to AlexaFluor 647 (albumin-AF647). CD144 (VE-cadherin) and AlexaFluor 647 are shown in the 488 and 647 nm channels, respectively. Hoechst nuclear counterstain is overlaid in all images. Scale bar: 200 μm. (B) Representative flow cytometry plots for isotype control, PBS- and Dox-treated cells from the fluorescent albumin accumulation assay showing gating of the CD144 high (CD144hi) and CD144 low (CD144lo) subpopulations. (C) Quantification of albumin-AF647 accumulation in gated subpopulations incubated at 37 °C. Statistical comparisons shown between accumulation for each individual subpopulation. (D) Quantification of albumin-AF647 accumulation in all CD144-positive cells from each condition, with or without MβCD pretreatment. Conditions incubated at 37 °C as well as 4 °C control were included. Selected statistical comparisons are shown among PBS- or Dox-treated conditions in the presence and absence of MβCD-pretreatment. Statistical analyses for ( C ) and ( D ) were performed on raw MFI data; P-values < 0.05 by one-way ANOVA with post-hoc Tukey’s test

Article Snippet: Lentivirus encoding N1ICD -IRES- GFP (denoted N1ICD LV) and IRES- GFP negative control (denoted GFP LV) constructs were produced in HEK293TN cells (System Biosciences, Palo Alto, CA).

Techniques: Over Expression, Epifluorescence Microscopy, Cell Culture, Incubation, Flow Cytometry, Control

Journal: Fluids and Barriers of the CNS

Article Title: Notch signaling activation reduces vesicular endocytosis in human pluripotent stem cell-derived CNS-like endothelial cells

doi: 10.1186/s12987-025-00754-6

Figure Lengend Snippet: N1ICD overexpression reduces abundance of total and caveolae-associated vesicles: Transmission electron microscopy (TEM) analysis of PBS- or Dox-treated PB-TRE-N1ICD 10(+ CR2) hPSC-CECs. (A) Representative ultrastructural TEM images. Red arrowheads indicate locations of vesicles. Scale bar: 400 nm. (B) Quantification of vesicle density relative to total cell area for each image. (C) Quantification of vesicle count per diameter range across all 12 images for PBS- or Dox-treated conditions. (D) Representative immunolabeled TEM image of Nanogold particles bound to caveolin-1 in PBS-treated cells. Scale bar: 100 nm. (E) Quantification of area fraction occupied by Nanogold particles relative to total cell area. (F) Quantification of caveolin-1/Nanogold-associated vesicles relative to total cell area. For ( C ), ( E ), and ( F ), points represent 3 images measured per biological replicate for n = 4 replicates in each treatment condition. Horizontal bars indicate mean ± SD. Statistical analyses were performed on cell area-normalized counts for ( C ) and ( F ) and on area fraction for ( E ). All P-values < 0.05 by Student’s t -test

Article Snippet: Lentivirus encoding N1ICD -IRES- GFP (denoted N1ICD LV) and IRES- GFP negative control (denoted GFP LV) constructs were produced in HEK293TN cells (System Biosciences, Palo Alto, CA).

Techniques: Over Expression, Transmission Assay, Electron Microscopy, Immunolabeling

Journal: Cell Death & Disease

Article Title: A novel super-enhancer-driven lncRNA LINC00973 governs head and neck squamous cell carcinoma progression through EN2

doi: 10.1038/s41419-025-08380-8

Figure Lengend Snippet: a Schematic diagram outlining the experimental design to identify and characterize downstream signaling pathways modulated by EN2 in HNSCC. b Differential analyses of KEGG pathways were performed in Cal27 and HN6 cells with/without EN2 silencing and the results were represented as scatter plot (left panel) and heatmap (right panel). KEGG pathway scores were calculated by GSVA method. c Correlation analyses between LINC00973 expression and KEGG_NOTCH_SIGNALING_PATHWAY scores (GSVA method) in TCGA-HNSC, GSE186775 and GSE65858 datasets. d The transcription activity of NOTCH1, the core member of NOTCH pathway, was measured by NOTCH1 luciferase reporter assays in Cal27 and HN6 with EN2 manipulations. e The protein abundance of NOTCH1, N1ICD and HEY1 were determined by western blot in Cal27 and HN6 cells with EN2 knockdown. f The endogenous NOTCH1, N1ICD and HEY1 protein expression was detected in Cal27 and HN6 cells followed by LINC00973 siRNAs or HN30 cells with LINC00973 overexpression, respectively. g NOTCH1, N1ICD and HEY1 were determined by western blot in Cal27 and HN6 cells with/without LINC00973 knockdown coupled with/withoutEN2 overexpression. Cell proliferation, migration and invasion were significantly increased following LINC00973 overexpression but reduced by DAPT treatment as gauged by CCK-8 ( h ), wound healing ( i ) and Transwell invasion assays ( j ).Scale bar: 50 μm. k Representative H&E, IHC staining of FOSL1, EN2, and NOTCH1 as well as FISH staining of LINC00973, in serial sections of the same HNSCC clinical samples were shown. Scale bar: 50 μm. l The correlation among FOSL1, EN2 and NOTCH1 protein expression (IHC quantifications data) and LINC00973 RNA abundance (FISH signal intensity) were analyzed by using Spearman’s correlation. Data were presented as mean ± SD, ** P < 0.01, Student’s t test.

Article Snippet: Next, membranes were blocked with 5% non-fat dry milk and incubated overnight at 4 °C with the indicated primary antibodies: EN2 (1:500, CSB-PA007660LA01HU, Cusabio, China), N-cadherin (1:2000, #22018-1-AP, Proteintech, China), E-cadherin (1:20000, #20874-1-AP, Proteintech, China), Vimentin (1:20000, #10366-1-AP, Proteintech, China), GAPDH (1:50000, #60004-1-Ig, Proteintech, China), FOSL1 (1:1000, #5281, Cell signaling, USA), NOTCH1 (1:1000, #20687-1-AP, Proteintech, China), BRD4 (1:1000, #13440, Cell signaling, USA), HEY1 (1:1000, #19929-1-AP, Proteintech, China), N1ICD (1:1000, #4147, Cell signaling, USA) or Rpb1 (1:1000, #14958, Cell signaling, USA).

Techniques: Protein-Protein interactions, Expressing, Activity Assay, Luciferase, Quantitative Proteomics, Western Blot, Knockdown, Over Expression, Migration, CCK-8 Assay, Immunohistochemistry, Staining

Journal: The Journal of Clinical Investigation

Article Title: DLL4 + neutrophils promote Notch1-mediated endothelial PANoptosis to exacerbate acute lung injury in sepsis

doi: 10.1172/JCI194310

Figure Lengend Snippet: ( A ) Computational modeling for the protein-protein interaction analysis between Notch1 extracellular domain and DLL4. Notch1 is shown in green, DLL4 in blue, and the interaction between Notch1 and DLL4 in purple. ( B ) Computational modeling was used to design Notch1-DLL4 inhibitor (NDI) derived from the Notch1 sequence that binds to DLL4. DLL4 is shown in blue, NDI in red. ( C and D ) BIAcore analysis was conducted to determine the binding affinity ( K D value) of DLL4 to Notch1 and the inhibitory effect of NDI on this interaction. Wild-type PVECs (0.5 × 10 6 ) were cocultured with sorted DLL4 + neutrophils (0.5 × 10 6 ) or DLL4 – neutrophils (0.5 × 10 6 ), then treated with either 10 μM scramble or NDI. PVECs (0.5 × 10 6 ) were first treated with Notch1-knockdown plasmid or its negative control for 24 hours. These PVECs were then cocultured with sorted DLL4 + neutrophils (0.5 × 10 6 ). ( E ) After 16 hours, total RNA was extracted and ZBP1 expression was analyzed by RT-PCR. Experiments were performed at least 3 times, and all data were analyzed. Data are expressed as means ± SEM and were analyzed using 1-way ANOVA. n = 8–15 per group. ( F – I ) Western blot analysis was performed to measure protein levels of ZBP1, c-GSDMD, t-GSDMD, c-caspase-3, t-caspase-3, p-MLKL, and MLKL in PVECs. Pyroptosis, apoptosis, and necroptosis were quantified as c/t GSDMD, c/t caspase-3, and p-MLKL/MLKL. n = 6–14 per group. Data are representative of 3 independent experiments. Data are expressed as means ± SEM and were analyzed using 1-way ANOVA. * P < 0.05 vs. PBS. # P < 0.05 vs. DLL4 + neutrophils/scramble. Con, Notch1-knockdown plasmid negative control–treated PVECs; Ko, Notch1-knockdown plasmid–treated PVECs.

Article Snippet: The ZBP1 antibody (catalog PA5-20455; 1:1,000) used in this study was obtained from Thermo Fisher Scientific, and the c-GSDMD (catalog 10137S; 1:250), GSDMD (catalog 39754S; 1:1000), c-caspase-3 (catalog 9661L; 1:250), caspase-3 (catalog 9662S; 1:1,000), p-MLKL (catalog 37333S; 1:250), MLKL (catalog 37705S; 1:1,000), N1ICD (c-Notch1) (catalog 4147S; 1:250), and Notch1 (catalog 3608S; 1:1,000) antibodies were obtained from Cell Signaling Technology.

Techniques: Derivative Assay, Sequencing, Binding Assay, Knockdown, Plasmid Preparation, Negative Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: DLL4 + neutrophils promote Notch1-mediated endothelial PANoptosis to exacerbate acute lung injury in sepsis

doi: 10.1172/JCI194310

Figure Lengend Snippet: DLL4 + neutrophils induce pulmonary endothelial cell PANoptosis via the Notch1-DLL4 pathway in sepsis. eCIRP induces neutrophils to express DLL4, and these DLL4 + neutrophils interact with PVECs through the Notch1-DLL4 pathway. This interaction triggers endothelial PANoptosis, driving inflammation and ALI in sepsis. NDI effectively inhibits the Notch1-DLL4 interaction, reducing endothelial PANoptosis and ALI, thereby improving survival outcomes in sepsis. The schema was created in BioRender (Murao A, 2025, https://BioRender.com/y914a2i ).

Article Snippet: The ZBP1 antibody (catalog PA5-20455; 1:1,000) used in this study was obtained from Thermo Fisher Scientific, and the c-GSDMD (catalog 10137S; 1:250), GSDMD (catalog 39754S; 1:1000), c-caspase-3 (catalog 9661L; 1:250), caspase-3 (catalog 9662S; 1:1,000), p-MLKL (catalog 37333S; 1:250), MLKL (catalog 37705S; 1:1,000), N1ICD (c-Notch1) (catalog 4147S; 1:250), and Notch1 (catalog 3608S; 1:1,000) antibodies were obtained from Cell Signaling Technology.

Techniques:

Journal: Advanced Science

Article Title: Targeting the Notch1‐YY1‐ICAM1 Signaling Axis Enhances the Efficacy of Immunotherapy in HCC by Activating CD8 + T‐Cell‐Driven Cancer Cell Pyroptosis

doi: 10.1002/advs.202512845

Figure Lengend Snippet: High Notch1 expression indicates poor therapeutic efficacy of immune checkpoint inhibitors and poor outcomes in HCC patients. A) Therapeutic response to anti‐PD‐1/PD‐L1 monoclonal antibodies in two representative HCC patients with high or low N1ICD expression. Representative CT images, immunohistochemical staining of N1ICD and PD‐L1, serum AFP levels, and PFS data are shown. The tumor border is marked by red lines in the MR images. The red arrow indicates the timing of anti‐PD‐1/PD‐L1 treatment in HCC patients. B) High N1ICD expression correlated with poor progression‐free survival in HCC patients after receiving adjuvant anti‐PD‐L1/PD‐1 treatment ( n = 34 HCC patients; cohort 1). C) Therapeutic response to immunotherapy in HCC patients with low or high N1ICD expression according to the mRECIST guidelines according to the CT/MRI results. CR, complete response; PR, partial response; PD, progressive disease; SD, stable disease. D) Immunotherapeutic response in HCC patients with high or low N1ICD ( n = 34 HCC patients; cohort 1). Each sample on the violin plots represents individual patient data (NR = nontonder, R = responder). E) Western blot analysis confirmed the knockdown of N1ICD in Hepa‐1‐6 cells. F) C57BL/6 mice were orthotopically injected with Hepa‐1‐6 shN1ICD or Hepa‐1‐6 scr . The liver tumor‐bearing mice were intraperitoneally injected with 5 mg kg −1 PD‐L1 antibody or IgG control antibody on day 7 and treated twice a week for up to 2 weeks ( n = 5 mice per group). A representative gross tumor image from each group is shown. The bar chart shows the final tumor volume of each group. The means ± SEMs are given. ** p < 0.01, *** p < 0.001, **** p < 0.0001. B) Log‐rank test. D) Student's t ‐test. The scale bars in (A) represent 2 cm (white), A) 100 µm (black), and F) 1 cm.

Article Snippet: After lysis, an ultrasonic disruptor (Bioruptor PLUS, Canada) was used to break the DNA genome into approximately 400–800‐bp protein‒DNA complexes, which were then incubated with N1ICD antibody (Cell Signaling Technology, Cat. no. 3608s), YY1 antibody (Cell Signaling Technology, Cat. no. 46395S) or IgG control antibody (Cell Signaling Technology, Cat. no. 3900S) as a control.

Techniques: Expressing, Drug discovery, Clinical Proteomics, Bioprocessing, Immunohistochemical staining, Staining, Adjuvant, Western Blot, Knockdown, Injection, Control

Journal: Advanced Science

Article Title: Targeting the Notch1‐YY1‐ICAM1 Signaling Axis Enhances the Efficacy of Immunotherapy in HCC by Activating CD8 + T‐Cell‐Driven Cancer Cell Pyroptosis

doi: 10.1002/advs.202512845

Figure Lengend Snippet: N1ICD expression in cancer cells regulates human cytotoxic T lymphocyte‐mediated killing in vitro and in vivo. A) Both Western blot and RT‒PCR confirmed the knockdown of N1ICD in Huh7 cells. B) Preparation process of cancer cell lysate‐pulsed dendritic cells primed with CD8 + T cells (created with BioRender.com). C–E) LDH release assay of HepG2 N1ICD /HepG2 ctrl cells, Huh7 shN1ICD /Huh7 scr cells or MHCC‐97H shN1ICD /MHCC‐97H scr cells after co‐culture with tumor‐specific CD8 + T cells at different E/T ratios as indicated ( n = 3 independent experiments). F) Flow cytometry analysis of CD107a expression on tumor‐specific CD8 + T cells after co‐culture with HepG2 N1ICD /HepG2 ctrl cells, Huh7 shN1ICD /Huh7 scr cells or MHCC‐97H shN1ICD /MHCC‐97H scr cells (E/T ratio: 10:1) as indicated. G) Bar chart showing the ratio of CD8 + CD107a + T cells in each group. H) LDH release assays of Huh7 shN1ICD /MHCC‐97H shN1ICD cells after co‐culture with tumor‐specific CD8 + T cells (E/T ratio: 10:1) in the presence of the DMSO solvent control or BMS‐1 (10 µ m ). I) NOD/SCID mice were subcutaneously injected with 3 × 10 6 Huh7 shN1ICD cells, which were treated with either an anti‐PD‐L1 (aPD‐L1) or anti‐IgG (IgG) antibody together with adoptive cell transfer (ACT: tumor‐specific CD8 + T cells). A representative gross tumor image from each group is shown. J) Representative images of H&E‐stained sections from each group are shown. K) Tumor growth curves of Huh7 shN1ICD cells after treatment with tumor‐specific CD8 + T cells in the presence of aPD‐L1 or IgG ( n = 5 mice per group). L) Bar chart showing the final tumor volume in each treatment group ( n = 3 mice per group). The means ± SEMs are given. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. A,C–E,G,H,K,L) Student's t ‐test. The scale bars in (I) represent 1 cm, and those in (J) represent 50 µm.

Article Snippet: After lysis, an ultrasonic disruptor (Bioruptor PLUS, Canada) was used to break the DNA genome into approximately 400–800‐bp protein‒DNA complexes, which were then incubated with N1ICD antibody (Cell Signaling Technology, Cat. no. 3608s), YY1 antibody (Cell Signaling Technology, Cat. no. 46395S) or IgG control antibody (Cell Signaling Technology, Cat. no. 3900S) as a control.

Techniques: Expressing, In Vitro, In Vivo, Western Blot, Knockdown, Lactate Dehydrogenase Assay, Co-Culture Assay, Flow Cytometry, Solvent, Control, Injection, Staining

Journal: Advanced Science

Article Title: Targeting the Notch1‐YY1‐ICAM1 Signaling Axis Enhances the Efficacy of Immunotherapy in HCC by Activating CD8 + T‐Cell‐Driven Cancer Cell Pyroptosis

doi: 10.1002/advs.202512845

Figure Lengend Snippet: N1ICD expression in cancer cells prohibits tumor‐specific CD8 + T‐cell‐secreted granzyme‐mediated GSDMB‐driven cancer cell pyroptosis. A) Representative high‐throughput automated confocal images of HepG2 N1ICD /HepG2 ctrl cells after co‐culture with tumor‐specific CD8 + T cells (E:T ratio 10:1) are shown. The white arrows indicate pyroptotic cells. B) LDH release assay of HepG2 N1ICD /HepG2 ctrl cells after co‐culture with tumor‐specific CD8 + T cells ( n = 3 independent experiments). C) ELISAs of GZMA secretion in the culture medium of tumor‐specific CD8 + T cells after co‐culture with HepG2 N1ICD /HepG2 ctrl cells. D) Representative high‐throughput automated confocal images of Huh7 shN1ICD /Huh7 scr cells after co‐culture with tumor‐specific CD8 + T cells (E:T ratio 10:1) are shown. E) LDH release assay of Huh7 shN1ICD /Huh7 scr cells after co‐culture with tumor‐specific CD8 + T cells ( n = 3 independent experiments). F) ELISAs of GZMA secretion in the culture medium of tumor‐specific CD8 + T cells after co‐culture with Huh7 shN1ICD /Huh7 scr cells. G) Representative high‐throughput automated confocal images of cancer cells after co‐culture with tumor‐specific CD8 + T cells in the presence of the granzyme inhibitor EGTA or CD8 + T cells pretreated with the pangranzyme inhibitor DCI are shown. H) LDH release of cancer cells after co‐culture with either tumor‐specific CD8 + T cells in the presence of the granzyme inhibitor EGTA or tumor‐specific CD8 + T cells pretreated with the pangranzyme inhibitor DCI as indicated. I) ELISAs of GZMA secretion in the culture medium of tumor‐specific CD8 + T cells after co‐culture with Huh7 shN1ICD /Huh scr cells. J) Both Western blot and RT‒qPCR confirmed the knockdown of GSDMB expression in Huh7 shN1ICD cells. K) Representative high‐throughput automated confocal images of GSDMB‐silenced Huh7 shN1ICD cells/scramble‐transfected cells after co‐culture with tumor‐specific T cells are shown. L) LDH assays of GSDMB‐silenced Huh7 shN1ICD cells/scramble‐transfected Huh7shN1ICD cells. ( n = 3 independent experiments). M) Both Western blot and RT‒qPCR confirmed the overexpression of GSDMB in Hepa1‐6 cells. N) LDH release assay of Hepa1‐6 shN1ICD cells/Hepa1‐6 scr cells after co‐culture with tumor‐specific CD8 + T cells at different E/T ratios as indicated ( n = 3 independent experiments). The means ± SEMs are given. ** p < 0.01, *** p < 0.001, **** p < 0.0001. B,C,E,F,H–J,L–N) Student's t ‐test. The scale bars in (A,D,G,K) represent 50 µm.

Article Snippet: After lysis, an ultrasonic disruptor (Bioruptor PLUS, Canada) was used to break the DNA genome into approximately 400–800‐bp protein‒DNA complexes, which were then incubated with N1ICD antibody (Cell Signaling Technology, Cat. no. 3608s), YY1 antibody (Cell Signaling Technology, Cat. no. 46395S) or IgG control antibody (Cell Signaling Technology, Cat. no. 3900S) as a control.

Techniques: Expressing, High Throughput Screening Assay, Co-Culture Assay, Lactate Dehydrogenase Assay, Western Blot, Knockdown, Transfection, Over Expression

Journal: Advanced Science

Article Title: Targeting the Notch1‐YY1‐ICAM1 Signaling Axis Enhances the Efficacy of Immunotherapy in HCC by Activating CD8 + T‐Cell‐Driven Cancer Cell Pyroptosis

doi: 10.1002/advs.202512845

Figure Lengend Snippet: N1ICD regulates ICAM expression in cancer cells to determine the immunotherapeutic response in HCC. A) Heatmap showing the RNA sequencing results of N1ICD‐depleted Huh7 cells compared with scramble‐transfected cells. B) GO enrichment analysis revealed a significant difference in several biological processes between Huh7 shN1ICD cells and Huh7 scr cells. C) Venn diagram analysis of the GO biological processes revealed that the expression of IL18R1, CD47, LGALS3, ANXA1, TNFSF4, ZP3, ICAM1, IL7R, CD81, FUT7, MYB, IL18, PRKCZ, FCER1G, HLA‐DMB, and F2RL1 was altered in Huh7 shN1ICD cells compared with Huh7 scr cells . D) RT‒qPCR analysis of altered gene expression in HepG2 N1ICD /HepG2 ctrl cells, Huh7 shN1ICD /Huh7 scr cells, and MHCC‐97H shN1ICD /MHCC‐97H scr cells ( n = 3 independent experiments). F) Western blot analysis of ICAM1 expression in HepG2 N1ICD /HepG2 ctrl cells or Huh7 shN1ICD /Huh7 scr cells. G) Low ICAM1 expression correlated with poor progression‐free survival in HCC patients who received anti‐PD‐1/PD‐L1 antibody treatment ( n = 34 HCC patients; cohort 1). H) Therapeutic response to immunotherapy in HCC patients with low or high ICAM1 expression according to the mRECIST guidelines. I) Immunotherapeutic response in HCC patients with high or low ICAM1 ( n = 34 HCC patients, cohort 1). Each sample on the violin plots represents individual patient data. J) Representative high‐throughput automated confocal images and LDH release assay of ICAM1‐overexpressing Huh7 (Huh7 ICAM1 )/control empty vector‐transfected (Huh7 ctrl ) cells after co‐culture with tumor‐specific CD8 + T cells. K) At 7 days after orthotopic Hepa‐1‐6 N1ICD cell injection, the tumor‐bearing mice were treated with the PEI‐ICAM1 complex (2 mg kg −1 , i.v.) every three days for up to 7 days together with IgG or aPD‐L1 (5 mg kg −1 , i.p.) every 3 days for a total of 3 times. A representative gross tumor image from each treatment group is shown. The bar chart shows the final tumor volume in each group ( n = 5 mice per group). The means ± SEMs are given. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. C–E,I–K) Student's t test. (G) Log‐rank test. Scale bars in (L) represent 50 µm, (K) 1 cm.

Article Snippet: After lysis, an ultrasonic disruptor (Bioruptor PLUS, Canada) was used to break the DNA genome into approximately 400–800‐bp protein‒DNA complexes, which were then incubated with N1ICD antibody (Cell Signaling Technology, Cat. no. 3608s), YY1 antibody (Cell Signaling Technology, Cat. no. 46395S) or IgG control antibody (Cell Signaling Technology, Cat. no. 3900S) as a control.

Techniques: Expressing, RNA Sequencing, Transfection, Gene Expression, Western Blot, Clinical Proteomics, High Throughput Screening Assay, Lactate Dehydrogenase Assay, Control, Plasmid Preparation, Co-Culture Assay, Injection

Journal: Advanced Science

Article Title: Targeting the Notch1‐YY1‐ICAM1 Signaling Axis Enhances the Efficacy of Immunotherapy in HCC by Activating CD8 + T‐Cell‐Driven Cancer Cell Pyroptosis

doi: 10.1002/advs.202512845

Figure Lengend Snippet: Cancer cell‐N1ICD downregulates ICAM1 expression to prohibit tumor‐specific CD8 + T‐cell cytotoxicity and mediate cancer cell pyroptosis. A) LDH release assay of Huh7 shN1ICD /MHCC‐97H shN1ICD cells after treatment with tumor‐specific CD8 + T cells at different E/T ratios in the presence of the IgG control or ICAM‐1 neutralizing antibody. B) Both Western blot and RT‒qPCR confirmed the knockdown of ICAM1 expression in Huh7 shN1ICD cells. C) Representative high‐throughput automated confocal images of ICAM1‐silenced Huh7 shN1ICD cells/scramble‐transfected cells after co‐culture with tumor‐specific CD8 + T cells are shown. D) LDH release assay of ICAM1‐silenced Huh7 shN1ICD cells/scramble‐transfected cells after co‐culture with tumor‐specific CD8 + T cells. E) ELISAs of GZMA expression in the culture medium of ICAM1‐silenced Huh7 shN1ICD cells/scramble‐transfected cells after co‐culture with tumor‐specific CD8 + T cells. F) Both Western blot and RT‒qPCR confirmed the expression of ICAM1 in HepG2 N1ICD cells. G) Representative high‐throughput automated confocal images of stable ICAM1‐expressing HepG2 N1ICD cells and control cells after treatment with tumor‐specific CD8 + T cells. H) LDH release assay and GZMA ELISA of stable ICAM1‐expressing HepG2 N1ICD cells and control cells after treatment with tumor‐specific CD8 + T cells ( n = 3 independent experiments). I) Venn diagram analysis showing the transcription factor‐binding site prediction of the ICAM1 promoter via three different online databases (i.e., CistromeDB, PROMO, and hTFtarget (liver)). J–L) Both RT‒PCR and Western blot analysis revealed that YY1 expression was upregulated in HepG2 N1ICD cells compared with that in HepG2 ctrl cells, whereas YY1 expression was downregulated in Huh7 shN1ICD /MHCC‐97H shN1ICD cells compared with that in scramble‐transfected cells. The means ± SEMs are given. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. A,B,D–F,H,J–L) Student's t ‐test. The scale bars in (C,G) represent 50 µm.

Article Snippet: After lysis, an ultrasonic disruptor (Bioruptor PLUS, Canada) was used to break the DNA genome into approximately 400–800‐bp protein‒DNA complexes, which were then incubated with N1ICD antibody (Cell Signaling Technology, Cat. no. 3608s), YY1 antibody (Cell Signaling Technology, Cat. no. 46395S) or IgG control antibody (Cell Signaling Technology, Cat. no. 3900S) as a control.

Techniques: Expressing, Lactate Dehydrogenase Assay, Control, Western Blot, Knockdown, High Throughput Screening Assay, Transfection, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Advanced Science

Article Title: Targeting the Notch1‐YY1‐ICAM1 Signaling Axis Enhances the Efficacy of Immunotherapy in HCC by Activating CD8 + T‐Cell‐Driven Cancer Cell Pyroptosis

doi: 10.1002/advs.202512845

Figure Lengend Snippet: N1ICD transcriptionally upregulates the expression of the transcriptional repressor YY1 to repress ICAM1 levels in HCC cells. A) High expression of YY1 was correlated with poor progression‐free survival, increased recurrence, and a worse immunotherapeutic response in HCC patients after treatment with adjuvant PD‐1/PD‐L1 antibody therapy ( n = 34 HCC patients, cohort 1). B) Stacked bar showing the therapeutic response to immunotherapy in HCC patients with low or high YY1 expression according to the mRECIST guidelines. C) Violin plot showing the expression level of YY1 in responders and nonresponders. Each sample on the violin plots represents individual patient data ( n = 34 HCC patients, cohort 1). D) Representative immunohistochemistry images of YY1, ICAM1, and Notch1 staining in tumor sections derived from HCC patients with high or low Notch1 expression are shown. E) In our HCC cohort 1 ( n = 34), the expression of Notch1 and ICAM1 was negatively correlated (left), the expression of YY1 and Notch1 was positively correlated (middle), and the expression of YY1 and ICAM1 was negatively correlated (right). F) Depletion of YY1 by siRNA in HepG2 N1ICD cells enhanced tumor‐specific CD8 + T‐cell‐mediated cancer cell pyroptosis and cytotoxicity ( n = 3 independent experiments). G) LDH release assay of stable N1ICD‐expressing HepG2 cells after transfection with YY1‐targeting siRNA after treatment with tumor‐specific CD8 + T cells in the presence of DMSO or BMS‐1 ( n = 3 independent experiments). H) Overexpression of YY1 in Huh7 shN1ICD cells inhibited CD8 + T‐cell‐mediated cancer cell pyroptosis ( n = 3 independent experiments). I) Schematic diagram showing the putative N1ICD binding sites in the human YY1 promoter. J) Luciferase assay of HepG2 N1ICD /HepG2 ctrl cells transfected with a luciferase reporter containing either the full‐length WT YY1 promoter or the mutated YY1 promoter sequence. K) ChIP assays revealed increased binding of N1ICD to its 2 nd putative binding site on the YY1 promoter in HepG2 N1ICD cells compared with that in HepG2 ctrl cells. L) Schematic diagram showing the putative YY1 binding sites in the human ICAM1 promoter. M) Luciferase assay of Huh7 shN1ICD cells transfected with a luciferase reporter containing either a full‐length ICAM1 promoter or mutated ICAM1 promoter sequence in the presence of the YY1 overexpression vector or empty control vector (ctrl). N) ChIP assays revealed increased binding of YY1 to its 1 st putative binding site on the ICAM1 promoter in Huh7 shN1ICD cells transiently transfected with the YY1 overexpression vector compared with empty vector‐transfected cells ( n = 3 independent experiments). The means ± SEMs are given. ns, nonsignificant difference. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. A) Log‐rank test. E) Spearman correlation study. C,F–H,J,K,M,N) Student's t ‐test. Scale bars in (F, H) represent 50 µm, D) 100 µm.

Article Snippet: After lysis, an ultrasonic disruptor (Bioruptor PLUS, Canada) was used to break the DNA genome into approximately 400–800‐bp protein‒DNA complexes, which were then incubated with N1ICD antibody (Cell Signaling Technology, Cat. no. 3608s), YY1 antibody (Cell Signaling Technology, Cat. no. 46395S) or IgG control antibody (Cell Signaling Technology, Cat. no. 3900S) as a control.

Techniques: Expressing, Adjuvant, Clinical Proteomics, Immunohistochemistry, Staining, Derivative Assay, Lactate Dehydrogenase Assay, Transfection, Over Expression, Binding Assay, Luciferase, Sequencing, Plasmid Preparation, Control

Journal: Advanced Science

Article Title: Targeting the Notch1‐YY1‐ICAM1 Signaling Axis Enhances the Efficacy of Immunotherapy in HCC by Activating CD8 + T‐Cell‐Driven Cancer Cell Pyroptosis

doi: 10.1002/advs.202512845

Figure Lengend Snippet: Combined treatment with the PEI‐siYY1 complex and PD‐L1 antibody significantly repressed orthotopic HCC tumor growth without causing any adverse side effects. A) Schematic diagram of the combined treatment of PD‐L1 monoclonal antibody and DAPT or PEI‐siYY1 complex in the HCC mouse orthotopic model (created with BioRender.com). Seven days after orthotopic Hepa‐1‐6 cell injection, the tumor‐bearing mice were treated with either DAPT (10 mg kg −1 , s.c.) for 7 consecutive days or the PEI‐siYY1 complex (2 mg kg −1 , i.v.) every 3 days for up to 7 days together with the IgG control or PD‐L1 antibody (aPD‐L1) (5 mg kg −1 , i.p.) every 3 days for up to 3 times. Tumor growth was monitored via in vivo ultrasound imaging every 3 days for a total of 3 days ( n = 5 mice per group). B,C) Representative gross Hepa1‐6 N1ICD (B)‐ or Hepa1‐6 shN1ICD (C)‐derived tumor images from each treatment group are shown (left), and the final tumor volumes are shown in a bar chart (right). D) Representative images of immunohistochemical staining for YY1 in the heart, lung, spleen, and kidney. E) Representative ultrasound scanning images (left), gross tumor images (middle), and final tumor volume quantification (right) of Hepa1‐6 orthotopic tumor models treated with DAPT combined with an anti‐PD‐L1 antibody (or solvent control) are shown ( n = 5 mice per group). F) Representative ultrasound scanning images (left), gross tumor images (middle), and final tumor volume quantification (right) of Hepa1‐6 orthotopic tumor models treated with PEI‐siYY1 combined with an anti‐PD‐L1 antibody (or solvent control) are shown ( n = 5 mice per group). The means ± SEMs are given. ** p < 0.01, *** p < 0.001, **** p < 0.0001. B,C,E,F) Student's t ‐test. The scale bars in (E) represent 1 mm, (B,C,E,F) 1 cm, and (D) 50 µm.

Article Snippet: After lysis, an ultrasonic disruptor (Bioruptor PLUS, Canada) was used to break the DNA genome into approximately 400–800‐bp protein‒DNA complexes, which were then incubated with N1ICD antibody (Cell Signaling Technology, Cat. no. 3608s), YY1 antibody (Cell Signaling Technology, Cat. no. 46395S) or IgG control antibody (Cell Signaling Technology, Cat. no. 3900S) as a control.

Techniques: Injection, Control, In Vivo, Imaging, Derivative Assay, Immunohistochemical staining, Staining, Solvent