Journal: JID Innovations

Article Title: Spatiotemporal fluorescence imaging of microRNA activity in 3-D models of human epidermis reveals contribution of the Notch pathway in the regulation of miR-30a in aging skin

doi: 10.1016/j.xjidi.2025.100444

Figure Lengend Snippet: The inhibition of the Notch pathway increases miR-30a activity. (a ) Representative images of reconstructed epidermises obtained using RIFES miR-30a-3p or miR-30a-5p primary keratinocytes. The green fluorescence corresponds to GFP, and the blue fluorescence corresponds to nuclei labeled with DAPI. The limit of the epidermis is indicated by a dotted line. Bar = 50 mm. ( b ) Immunofluorescence labeling of Notch 1 (denoted as NICD) in a skin biopsy (female abdominal skin, young adult). The red fluorescence corresponds to Notch1, the green fluorescence corresponds to loricrin, and the blue fluorescence corresponds to nuclei labeled with DAPI. Bar = 50 mm. ( c ) Western blot analysis of Notch1 (denoted as NICD), cleaved Notch1, and actin in HPKs treated or not by DAPT. ( d ) HEY1 transcript relative expression analysis by qRT-PCR in HPKs treated or not by DAPT (mean ± SD, n = 3, ∗ P < .05 t -test P -value). ( e ) Response of the RIFES miR-30a-3p or -5p HPKs to DAPT treatment. The graph corresponds to the quantification of the GFP fluorescence by HCS after DAPT or control treatment (box plot with Tukey whiskers, n = 4, ∗ P < .05 t -test P -value). Representative images of cells are shown (Merge image: GFP plus DAPI). ( f ) Western blot analysis of GFP and actin expression in lentiRIFES/miR-30a-3pT or 5pT HPKs after DAPT treatment. ( g ) Reconstructed epidermises were obtained using lentiRIFES/miR-30a-3pT or 5pT keratinocytes. The RHEs were treated or not by the DAPT compound. Representative images of GFP fluorescence or K10 immunofluorescence are shown. The limit of the epidermis is indicated by a dotted line. Bar = 50 mm. A small area of the images surrounded by a frame is shown at higher magnification. ( h ) miR-30a-3p or miR-30a-5p transcript expression analysis by qRT-PCR in HPKs treated or not by DAPT (mean ± SD, n = 3, ∗ P < .05 t -test P -value). HCS, high-content screening; HPK, human primary keratinocyte; K10, keratin 10; RHE, reconstructed human epidermis.

Article Snippet: Membranes were blocked with 10% nonfat dry milk for 1 hour and incubated with primary antibodies against NOTCH 1 (number 3608, Cell Signaling Technology), cleaved NOTCH-1 (number 4147, Cell Signaling Technology), c-MYC, or β-actin (ab8227, Abcam) overnight at 4 °C.

Techniques: Inhibition, Activity Assay, Fluorescence, Labeling, Immunofluorescence, Western Blot, Expressing, Quantitative RT-PCR, Control, High Content Screening

Journal: Scientific Reports

Article Title: Caveolin-1 modulates Notch transcriptional activity during in vitro respiratory multiciliated cell maturation

doi: 10.1038/s41598-026-40201-6

Figure Lengend Snippet: Analysis of Notch1 Expression and Subcellular Distribution in Luc-KD and Cav1-KD Cells. ( a ) Maximal projection of confocal images for Notch1 (in yellow), and nucleus (in blue) in Luc-KD and Cav1-KD cells in ALI 6. ( b ) Western blot images of Luc-KD and Cav1-KD cells showing Notch 1 (full-length at 300 kDa and cleaved at 120 kDa) and E-Cadherin expression. The lower Western blot image of Notch1 (300 kDa) in the panel shows a contrast-enhanced version of Notch1 region of interest (ROI) amplified to the entire image, derived from the original image with lower contrast shown in the upper part of the panel. Each condition was tested in triplicate (L1, L2, and L3 for each genotype). Cell lysates (L) were derived from an independent cell culture. β-actin was used as a loading control. ( c – e ) Relative protein expression levels quantification of Notch1 300 kDa ( c ), Notch1 120 kDa ( d ) and E-Cadherin ( e ). Mean and standard deviation as error bars were plotted, n = 3 independent lysates per group. ( f ) Analyses of Notch 1 full-length and processed forms (TMD+NICD and NICD) subcellular distribution in the cytosol (Cyt.), membrane (Mem.), and chromatin (Chr.) in Luc-KD and Cav1-KD cells. Cell fractionation and gradient SDS-Gels were used for improved resolution. ( g ) Relative protein expression quantification of Notch 1 subcellular distribution in Luc-KD and Cav1-KD cells. Mean and standard deviation as error bars were plotted, n = 4 independent lysates per group. ( h ) Proposed working model of the mechanism by which Cav-1 regulates BSC differentiation, involving differential NICD binding capacity to chromatin together with other partners. Scale bar in panel a represent 20 μm. p-values in all conditions were obtained using two-tailed t-test (*** represents p < 0.001, ** represents p < 0.01 and n.s. means no significative differences).

Article Snippet: The remaining chromatin was diluted, precleared with protein A/G Sepharose beads, and immunoprecipitated overnight at 4 °C using an anti-NICD antibody (Cell Signaling, #4147S, 2.5 μL of antibody/10 μg of chromatin).

Techniques: Expressing, Western Blot, Amplification, Derivative Assay, Cell Culture, Control, Standard Deviation, Membrane, Cell Fractionation, Binding Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: Caveolin-1 modulates Notch transcriptional activity during in vitro respiratory multiciliated cell maturation

doi: 10.1038/s41598-026-40201-6

Figure Lengend Snippet: Analysis of Notch2 Expression and Subcellular Distribution in Luc-KD and Cav1-KD Cells. ( a ) Maximal projection of confocal images for Notch2 (in green), and nucleus (in blue) in Luc-KD and Cav1-KD cells in ALI 6. ( b ) Western blot images of Luc-KD and Cav1-KD cells showing Notch 2 (full-length at 300 kDa and cleaved at 110 kDa) and c-Myb expression. Each condition was tested in triplicate (L1, L2, and L3 for each genotype). Cell lysates (L) were derived from an independent cell culture. GAPDH was used as a loading control. ( c – e ) Relative protein expression levels quantification of Notch2 300 kDa ( c ), Notch2 120 kDa ( d ) and c-Myb ( e ). Mean and standard deviation as error bars were plotted, n = 3 independent lysates per group. ( f ) Analyses of Notch 2 full-length and processed forms (TMD+NICD and NICD) subcellular distribution in the cytosol (Cyt.), membrane (Mem.), and chromatin (Chr.) in Luc-KD and Cav1-KD cells. Cell fractionation and gradient SDS-Gels were used for improved resolution. ( g ) Relative protein expression quantification of Notch 2 subcellular distribution in Luc-KD and Cav1-KD cells. Mean and standard deviation as error bars were plotted, n = 4 independent lysates per group. Scale bar in panel a represent 20 μm. p-values in all conditions were obtained using two-tailed t-test (**represents p < 0.01, *represents p < 0.05 and n.s. means no significative differences).

Article Snippet: The remaining chromatin was diluted, precleared with protein A/G Sepharose beads, and immunoprecipitated overnight at 4 °C using an anti-NICD antibody (Cell Signaling, #4147S, 2.5 μL of antibody/10 μg of chromatin).

Techniques: Expressing, Western Blot, Derivative Assay, Cell Culture, Control, Standard Deviation, Membrane, Cell Fractionation, Two Tailed Test

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

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