thle2  (ATCC)

Journal: Frontiers in Pharmacology

Article Title: Ultrafine garlic powder alleviates non-alcoholic steatohepatitis by inhibiting hepatocyte ferroptosis and modulating ERK-dependent oxidative stress

doi: 10.3389/fphar.2025.1711917

Figure Lengend Snippet: UGP ameliorates palmitatic acid-induced lipotoxic hepatocyte injury. Effects of palmitatic acid stimulation and Fer-1, UGP and TGP treatment of on human normal hepatocyte THLE2 cell viability was examined using CCK8 assay (A) and LDH assay (B) (n = 4). (C) Effects of palmitatic acid stimulation and Fer-1, UGP and TGP treatment of on THLE2 cell damage was determined by ALT assay (n = 4). (D) Cell death assay was performed using Annexin V/PI staining (n = 4). Annexin V + PI − , Annexin V + PI − , and Annexin V + PI + cells were considered dead cells. Representative fluorescent images and quantification of DCFDA (2′,7′-dichlorofluorescein diacetate) (E) and Mito-Tracker Red CMXRos (F) staining for ROS and mitochondrial membrane potential detection (scale bar = 100 μm, n = 4). (G) Flow cytometry analysis of lipid peroxidation by Liperfluo staining on THLE2 cells. (H) Representative immunofluorescent images of ACTA2 +ve and COL1A1 +ve cells (scale bar = 100 μm). Data are shown as box-and whisker with median (middle line), 25th–75th percentiles (box), and min-max values (whiskers), one-way ANOVA with Tukey’s correction. (ALT: Alanine transaminase; BSA:Bovine serum albumin; Fer-1: Ferrostatin-1; PAL: Palmitic acid; ROS: Reactive oxygen species; TGP: Traditional garlic powder; UGP:Ultrafine garlic powder).

Article Snippet: Human normal hepatocytes (THLE2) and hepatic stellate cells (HSCs) (LX-2) were purchased from the American Type Culture Collection (ATCC) and cultured in BEGM kit medium and RPMI medium supplemented with 10% FBS, respectively.

Techniques: CCK-8 Assay, Lactate Dehydrogenase Assay, Staining, Membrane, Flow Cytometry, Whisker Assay

Journal: Frontiers in Pharmacology

Article Title: Ultrafine garlic powder alleviates non-alcoholic steatohepatitis by inhibiting hepatocyte ferroptosis and modulating ERK-dependent oxidative stress

doi: 10.3389/fphar.2025.1711917

Figure Lengend Snippet: UGP significantly ameliorates erastin-induced hepatocyte ferroptosis. Effects of erastin stimulation and Fer-1, UGP and TGP treatment of on THLE2 cell viability was examined using CCK8 assay (A) and LDH assay (B) (n = 4). (C) Effects of erastin stimulation and Fer-1, UGP and TGP treatment of on THLE2 cell damage was determined by ALT assay (n = 4). (D) Cell death assay was performed using Annexin V/PI staining (n = 4). Annexin V + PI − , Annexin V + PI − , and Annexin V + PI + cells were considered dead cells. (E) Flow cytometry analysis of cellular Fe 2+ levels by FerroOrange staining on THLE2 cells. Representative fluorescent images and quantification of DCFDA (2′,7′-dichlorofluorescein diacetate) (F) and Mito-Tracker Red CMXRos (G) staining for ROS and mitochondrial membrane potential detection (scale bar = 100 μm, n = 4). (H) Flow cytometry analysis of lipid peroxidation by Liperfluo staining on THLE2 cells. Data are shown as box-and whisker with median (middle line), 25th-75th percentiles (box), and min-max values (whiskers), one-way ANOVA with Tukey’s correction. (ALT: Alanine transaminase; ERA: Erastin; Fer-1: Ferrostatin-1; ROS: Reactive oxygen species; TGP: Traditional garlic powder; UGP: Ultrafine garlic powder).

Article Snippet: Human normal hepatocytes (THLE2) and hepatic stellate cells (HSCs) (LX-2) were purchased from the American Type Culture Collection (ATCC) and cultured in BEGM kit medium and RPMI medium supplemented with 10% FBS, respectively.

Techniques: CCK-8 Assay, Lactate Dehydrogenase Assay, Staining, Flow Cytometry, Membrane, Whisker Assay

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