Journal: Cell Communication and Signaling : CCS

Article Title: Long non-coding RNA H19 transported by colorectal cancer small extracellular vesicles promotes alternative splicing in healthy hepatocytes: new insights on liver pre-metastatic niche formation

doi: 10.1186/s12964-026-02738-x

Figure Lengend Snippet: a qRT-PCR showing lncH19 levels inside CRC-sEVs and CRC_cells. b Representative images of western blot for the presence of RBFOX2 in the CRC-sEVs protein lysates. c Representative Image showing the proposed mechanism of lncH19 acting as shuttle RNA transferring RBFOX2 inside the EVs. d Representative confocal image of RNA in situ assay coupled with immunocytochemistry in the THLE2 treated for 3 h with the CRC-sEVs, lncH19 (red) and RBFOX2 (green). Scale bar:60 μm. The results reported in the graphs are the mean ± SD of three independent experiments. Statistical analyses were performed using Ordinary one-way ANOVA * p < 0.05, ** p < 0.01, *** p < 0.001. e Graphs showing the mean fluorescence intensity (MFI) relative to RBFOX2 (left) and lncH19 (right) of the different conditions

Article Snippet: The SV40 large T antigen-immortalized healthy human liver epithelial cell line THLE2 (ATCC, Manassas, VA) was cultured in Airway Epithelial Cell Basal Medium (ATCC, Manassas, VA) with the Bronchial Epithelial Cell Growth Kit (ATCC PCS-300–040, Manassas, VA) supplemented with 5 ng/ml epidermal growth factor (EGF), 70 ng/ml phosphoethanolamine, 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/ml streptomycin (Euroclone, UK) at 37 °C with 5% CO 2 .

Techniques: Quantitative RT-PCR, Western Blot, Transferring, In Situ, Immunocytochemistry, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Long non-coding RNA H19 transported by colorectal cancer small extracellular vesicles promotes alternative splicing in healthy hepatocytes: new insights on liver pre-metastatic niche formation

doi: 10.1186/s12964-026-02738-x

Figure Lengend Snippet: a Representative c onfocal micrographs of RNA in situ hybridisation in THLE2 treated for 18 h with the wt-CRC-sEVs and the sh-CRC-sEVs (lncH19 in red, Hoechst in blue). b MFI analysis obtained with NIS 1 A analysis software, and ( c ) qRT-PCR showing lncH19 levels in treated cells. ( d ) Representative confocal micrographs of immunocytochemistry for RBFOX2 in the THLE2 treated with wt- and sh- sEVs for 18 h and relative Scale bar: 60 μm ( e ) MFI analysis.The results reported in the graphs are the mean ± SD of three independent experiments. Statistical analyses were performed using Ordinary one-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The SV40 large T antigen-immortalized healthy human liver epithelial cell line THLE2 (ATCC, Manassas, VA) was cultured in Airway Epithelial Cell Basal Medium (ATCC, Manassas, VA) with the Bronchial Epithelial Cell Growth Kit (ATCC PCS-300–040, Manassas, VA) supplemented with 5 ng/ml epidermal growth factor (EGF), 70 ng/ml phosphoethanolamine, 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/ml streptomycin (Euroclone, UK) at 37 °C with 5% CO 2 .

Techniques: In Situ, Hybridization, Software, Quantitative RT-PCR, Immunocytochemistry

Journal: Cell Communication and Signaling : CCS

Article Title: Long non-coding RNA H19 transported by colorectal cancer small extracellular vesicles promotes alternative splicing in healthy hepatocytes: new insights on liver pre-metastatic niche formation

doi: 10.1186/s12964-026-02738-x

Figure Lengend Snippet: a qRT-PCR and ( b ) exon-specific qRT-PCR showing the expression levels of ENAH, CTTN and PARD3 genes in THLE2 treated with the wt- and sh-CRC-sEVs

Article Snippet: The SV40 large T antigen-immortalized healthy human liver epithelial cell line THLE2 (ATCC, Manassas, VA) was cultured in Airway Epithelial Cell Basal Medium (ATCC, Manassas, VA) with the Bronchial Epithelial Cell Growth Kit (ATCC PCS-300–040, Manassas, VA) supplemented with 5 ng/ml epidermal growth factor (EGF), 70 ng/ml phosphoethanolamine, 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/ml streptomycin (Euroclone, UK) at 37 °C with 5% CO 2 .

Techniques: Quantitative RT-PCR, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: Long non-coding RNA H19 transported by colorectal cancer small extracellular vesicles promotes alternative splicing in healthy hepatocytes: new insights on liver pre-metastatic niche formation

doi: 10.1186/s12964-026-02738-x

Figure Lengend Snippet: a Table showing the different miRNAs identified in the three different analyses (sponged by lncH19, involved in EMT and expressed by healthy liver tissue). b Venn Diagram from FunRich analysis of data obtained by the three indicated datasets. The square in figure indicates 4 from 11 miRNAs targeting PARD3 mRNA c qRT-PCR confirming the over-expression of lncH19 in the THLE2 and ( d ) the increase of PARD3 mRNA in the H19-overexpressing hepatocytes

Article Snippet: The SV40 large T antigen-immortalized healthy human liver epithelial cell line THLE2 (ATCC, Manassas, VA) was cultured in Airway Epithelial Cell Basal Medium (ATCC, Manassas, VA) with the Bronchial Epithelial Cell Growth Kit (ATCC PCS-300–040, Manassas, VA) supplemented with 5 ng/ml epidermal growth factor (EGF), 70 ng/ml phosphoethanolamine, 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/ml streptomycin (Euroclone, UK) at 37 °C with 5% CO 2 .

Techniques: Quantitative RT-PCR, Over Expression

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting NOTCH1-KEAP1 axis retards chronic liver injury and liver cancer progression via regulating stabilization of NRF2

doi: 10.1186/s13046-025-03488-3

Figure Lengend Snippet: Identification of potential indicators involved in chronic liver inflammation and hepatocellular carcinoma (HCC)-related biological processes. ( A ) Experimental design showing the protocol of identifying oxidative stress-associated indicators in response to time-course of 1% CCl 4 -, 10 mM DMN-induced human THLE2 cells and mouse primary hepatocytes, or in 6-weeks of CCl 4 (0.5 µL/g BW)- or DMN (0.5 mg/kg BW)-fed WT mice, or in liver samples from HCC, NASH, HBV, and HCV patients. ( B ) Venn diagram showing the Top 5 distinguishable expressed oxidative stress-associated candidates (i.e., NOTCH1, NRF2, GPX4, USP35 and ACSL4) in intersection of 4 separate samples data-set. ( C ) Waterfall streamgraph showing the relative mRNA expression profile of the 5 genes in the indicated groups. ( D ) Representative immunofluorescence images of NOTCH1 and HNF4A co-expression in lives of human samples with healthy control, HBV, HCV, NASH and HCC phenotype with fluorescence intensity measurement (magnification, 100×, n = 10 samples). ( E ) The flowchart showing the experimental procedure for the quantified proteome and protein interaction assay of NOTCH1. ( F ) Volcano plot indicating genes expression variation in human THLE2 cells after CCl 4 treatment. ( G ) Number of identified oxidative stress-related upregulated & downregulated sites. ( H ) A screening protocol to highlight assumed gene candidates. ( I ) The physiopathology and biological processes associated with metabolism of HBV, HCV, NASH, and HCC samples were found to differ from those of controls in a number of databases, including TCGA, ICGC, and the NCBI Gene Expression Omnibus (GEO) datasets (GEO: GSE225322 , GSE218332 , GSE282451 , GSE270921 , GSE267145 , GSE282660 , GSE205881 , and GSE290614 ). ( J ) The total number of differentially expressed genes that crossed over into different databases was tallied after gene differential expression analysis. ( K ) The molecular pathways influencing metabolism and the beginning of liver diseases are represented by seven upregulated DEGs. Data are presented as mean ± SEM. The associated experiments were performed independently at least three times. P < 0.05 indicates statistical significance

Article Snippet: Human THLE2 cell line (#CRL-2706), human HCC-related cell lines MHCC97H, SNU-182, SNU-398 and Hep3B were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Immunofluorescence, Control, Fluorescence, Protein Interaction Assay, Gene Expression, Quantitative Proteomics

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting NOTCH1-KEAP1 axis retards chronic liver injury and liver cancer progression via regulating stabilization of NRF2

doi: 10.1186/s13046-025-03488-3

Figure Lengend Snippet: NOTCH1 (NICD1) signaling is enhanced in liver samples of chronic liver injury patients and mice. ( A ) Relative gene expression analysis of NOTCH1 in liver specimens from patients with HBV, HCV, NASH and HCC pathological phenotype ( n = 12 samples). ( B ) Representative immunofluorescence images of NOTCH1 expression in liver samples of patients with HBV, HCV, NASH and HCC pathological phenotype (magnification, 100×, n = 10 samples). ( C ) Representative western blotting showing the NOTCH1 and NICD1 protein expression in liver samples isolated from patients with HBV, HCV, NASH and HCC pathological phenotype ( n = 12 samples). ( D , E ) Pearson’s r correlation analysis of AFP levels and NOTCH1 levels, and AST contents and NOTCH1 levels in patients ( n = 12 samples). ( F ) Multiple Pearson multiple correlation analysis for human subjects showing the comprehensive correlation between NOTCH1 protein expression levels and indicated parameter indexes ( n = 12 indices each parameter). Utilizing DMN- and CCl 4 -induced mice, NOTCH1 expression alterations were investigated in mouse models with liver samples. ( G ) Relative gene expression assay of NOTCH1 in livers of control group (NC) and DMN-treated group ( n = 10 samples). ( H ) Representative immunofluorescence images of NOTCH1 expression in liver samples of NC and DMN group ( n = 10 samples). ( I ) Western blotting analysis showing the NOTCH1, NICD1 and Hes1 expression in liver tissue isolated from indicated groups ( n = 4 samples). ( J ) Relative gene expression assay of NOTCH1 in livers of control group (NC) and CCl 4 -induced group ( n = 10 samples). ( K ) Representative immunofluorescence images of NOTCH1 expression in liver samples of NC and CCl 4 group ( n = 10 samples). ( L ) Western blotting analysis showing the NOTCH1, NICD1 and Hes1 expression in liver tissue isolated from NC or CCl 4 group ( n = 4 samples). ( M , N ) A dose-dependent rise in NOTCH1 gene expression levels and protein expression was detected in human THLE2 cells following 10 h of DMN incubation (10 µM, 20 µM, and 40 µM) ( n = 10 samples). ( O ) Representative immunofluorescence images of NOTCH1 and HNF4A co-expression in the indicated groups (magnification, 400×, n = 10 samples). Data are presented as mean ± SEM. The associated experiments were performed independently at least three times. P < 0.05 indicates statistical significance

Article Snippet: Human THLE2 cell line (#CRL-2706), human HCC-related cell lines MHCC97H, SNU-182, SNU-398 and Hep3B were purchased from the American Type Culture Collection (ATCC).

Techniques: Gene Expression, Immunofluorescence, Expressing, Western Blot, Isolation, Control, Incubation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting NOTCH1-KEAP1 axis retards chronic liver injury and liver cancer progression via regulating stabilization of NRF2

doi: 10.1186/s13046-025-03488-3

Figure Lengend Snippet: NOTCH1 interacts with and recruits KEAP1 in hepatocytes, leading to NRF2 polyubiquitination degradation under CCl 4 challenge. ( A ) THLE2 cells after transfection with Flag-NOTCH1 or the empty Vector were incubated with CCl 4 for 24 h simultaneously in the presence of the protein synthesis inhibitor cycloheximide (CHX; 50 µg/ml) for the indicated times (0 h, 3 h, 6 h, 9 h). Relative protein expression levels for NRF2 in transfected THLE2 cells after time-course treatment were quantified ( n = 4 per group). ( B ) Immunoblotting detection of Flag-NOTCH1 transfected THLE2 cells with/without CCl 4 (24 h), MG132 (20 µM, 12 h) and CHO (20 µM, 12 h) treatment ( n = 4 per group). ( C ) Left, the human THLE2 cells were transfected with the indicated plasmids. Anti-Nrf2 immunoprecipitates were analyzed by immunoblotting with anti-Ub antibody for the examination of ubiquitin-conjugated Nrf2. Right, the human THLE2 cells were transfected with the indicated plasmids. Anti-K48-Ub immunoprecipitates were analyzed by immunoblotting with anti-Ub antibody for the examination of ubiquitin-conjugated Nrf2. ( D ) The human THLE2 cells transfected with Ad NOTCH1 or AdGFP were incubated with CCl 4 for 24 h, and were then collected for qPCR analysis of NOTCH1 and KEAP1 ( n = 5 per group). ( E ) Western blot analysis for NOTCH1 and KEAP1 protein expression levels in 24 h of CCl 4 -treated THLE2 cells with or without Ad NOTCH1 transfection ( n = 4 per group). ( F ) Immunoprecipitation and western blot analysis indicating the binding of KEAP1 to NOTCH1 in human THLE2 cells transfected with Flag-NOTCH1 and HA-tagged KEAP1 (HA-KEAP1) under CCl 4 exposure. ( G ) Immunoprecipitation and immunoblotting assay showing the binding of KEAP1 to NOTCH1 in the liver samples of CCl 4 -treated mice; the IgG was served as a control. ( H ) Representative immunoblotting bands for GST precipitation showing NOTCH1-KEAP1 direct binding by treating purified NOTCH1-His with purified KEAP1-HA-GST or by treating KEAP1-His with purified NOTCH1-HA-GST in THLE2 cells. ( I ) Representative IF images showing NOTCH1 (green) and KEAP1 (red) in THLE2 cells challenged with/without CCl 4 for 24 h ( n = 5 independent biological replicates with 8 images per group). ( J ) Molecular docking analysis between NOTCH1 and KEAP1 protein. ( K ) Representative western blot of KEAP1 and NRF2 in THLE2 cells transfected with varying amounts of Flag-NOTCH1 with or without CCl 4 incubation for 24 h. ( L ) Luciferase assay of the fluorescence intensity of THLE2 cells transfected with increasing counts of Flag-NOTCH1 plasmids in response to HG treatment for 24 h ( n = 6 per group). ( M ) Western blot results for KEAP1 and NRF2 in the isolated hepatocytes from the shown groups ( n = 4 per group). ( N ) Schematic indicating full-length and truncated NOTCH1 (upper, left) and KEAP1 (upper, right) with representative Co-IP results (bottom) for the mapping analysis of the domains responsible for the NOTCH1-KEAP1 interaction in human THLE2 cells. ( O ) Western blots of NICD1, KEAP1, NRF2, GPX4, and p-NF-κB in human THLE2 cells transfected with AdGFP, Ad NOTCH1 (WT) or the ∆ANK NOTCH1/NICD1 variant at 24 h after CCl 4 treatment ( n = 3 per group). ( P ) DCF-DA staining, DHE staining, and Mito-SOX staining, and ( Q ) quantification for ROS production by respective staining were performed in human THLE2 cells transfected with AdGFP, Ad NOTCH1 (WT) or the ∆ANK NOTCH1/NICD1 variant in response to CCl 4 treatment for 24 h. ( R ) Lipid peroxidation was examined by C11-BODIPY in human THLE2 cells with AdGFP, Ad NOTCH1 (WT) or the ∆ANK NOTCH1/NICD1 variant under CCl 4 treatment for 24 h; red fluorescence represents the reduction form, while green fluorescence represents the oxidized form. ( S ) Mean intensity for the ratio of the oxidized form to reduced form was quantified related to C11-BODIPY staining ( n = 5 per group). ( T ) Mito-Tracker was used to examine the mitochondrial structure changes of THLE2 cells transfected with AdGFP, Ad NOTCH1 (WT) or the ∆ANK NOTCH1/NICD1 variant under CCl 4 treatment for 24 h, and the mean mitochondrial size was then quantified ( n = 6 per group). ( U ) Measurements for MDA levels, GSH contents, GSH/GSSG ratio, Fe 2+ levels and ATP levels in 24 h of CCl 4 -treated THLE2 cells with AdGFP, Ad NOTCH1 (WT) or the ∆ANK NOTCH1/NICD1 variant transfection ( n = 6 per group). ( V ) qPCR analysis for the mRNA expression levels of genes involved in oxidative stress and ferroptosis as shown in THLE2 cells transfected with AdGFP, Ad NOTCH1 (WT) or the ∆ANK NOTCH1/NICD1 variant after CCl 4 incubation for 24 h ( n = 4 in each group). ( W ) The mRNA expression levels of inflammatory genes were evaluated by qPCR in 24 h CCl 4 -incubated THLE2 cells transfected with AdGFP, Ad NOTCH1 (WT) or the ∆ANK NOTCH1/NICD1 variant ( n = 4 in each group). Data are presented as mean ± SEM. P < 0.05 indicates statistical significance. Two-tailed unpaired t -test was used to determine the p -values in ( A ), ( E ) and ( F ); Statistical comparisons in ( L ), ( M ), ( O ), ( Q ), ( S ) to ( W ) were performed using one-way ANOVA with a Tukey post-hoc analysis; the results in ( C ), ( G ) to ( I ), ( K ), and ( N ) were obtained from three independent experiments

Article Snippet: Human THLE2 cell line (#CRL-2706), human HCC-related cell lines MHCC97H, SNU-182, SNU-398 and Hep3B were purchased from the American Type Culture Collection (ATCC).

Techniques: Transfection, Plasmid Preparation, Incubation, Expressing, Western Blot, Ubiquitin Proteomics, Immunoprecipitation, Binding Assay, Control, Purification, Luciferase, Fluorescence, Isolation, Co-Immunoprecipitation Assay, Variant Assay, Staining, Two Tailed Test