Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: Hepatocyte TM4SF5-mediated downregulation of protein KEAP1 via their binding is irrelevant to NRF2. Hepatocytes including TM4SF5-null cell lines [SNU449 lacking endogenously and Huh7 TM4SF5-KO via CRISPR/Cas9-mediated KO , TM4SF5-expressing cell lines (Hep3B and Huh7 Control expressing endogenously), or primary hepatocytes from C57BL/6 male mice (G) were cultured at subconfluent conditions. Cells were transfected either transiently or stably with the relevant cDNAs. Additionally, they were exposed to TSAHC (a TM4SF5-specific chalcone inhibitor) for the indicated durations at varying concentrations (I) or transfected with shRNA targeting either a control sequence (shNS) or shNRF2 (target sequences #1 or #2, see Table ). Whole cell extracts were prepared using lysis buffer containing Triton X-100, Brij58 (H), or Triton X-100 alone (A-G and I-L). Following normalization, proteins were processed for (immuno)precipitation using streptavidin-agarose beads, anti-NRF2 antibody, or anti-KEAP1 antibody, prior to immunoblot analysis for designated molecules. Data shown are representative of three independent experiments. See also and S2.

Article Snippet: The primary antibodies used included TM4SF5-EC2 and KEAP1 (sc-365626, Santa Cruz Biotech.).

Techniques: Binding Assay, CRISPR, Expressing, Control, Cell Culture, Transfection, Stable Transfection, shRNA, Sequencing, Lysis, Immunoprecipitation, Western Blot

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: Correlations among TM4SF5, KEAP1, and CD36 levels following extracellular lipid treatment. (A-H) Subconfluent Huh7 (A, B, D, E, F, G, H, and I) and SNU449 (B, C) hepatocytes, either left unmodified or subjected to knockout (KO), were stably or transiently transfected with the specified cDNAs. For KEAP1 suppression, cells were treated with doxycycline (DOX) to induce silencing (G). In selected experiments, a lipid mixture (LM, 5%) was administered for 24 h. Following treatment, whole cell lysates (WCL) were prepared using lysis buffer containing Triton X-100. Lysates were quantified and subjected to immunoblotting (A, B, C, and G) or (immuno)precipitation (D, E, F, and H) with streptavidin-agarose beads, anti-Flag, or anti-KEAP1 antibodies, followed by immunoblotting for the indicated proteins. (I) Huh7 TM4SF5-KO cells complemented with mCherry-TM4SF5 WT were replated onto collagen I-precoated slide glasses and treated with either vehicle or lipid mixture prior to immunostaining and imaging. Scale bar: 100 μm. Data represent three independent experiments. See also .

Article Snippet: The primary antibodies used included TM4SF5-EC2 and KEAP1 (sc-365626, Santa Cruz Biotech.).

Techniques: Knock-Out, Stable Transfection, Transfection, Lysis, Western Blot, Immunoprecipitation, Immunostaining, Imaging

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: TM4SF5-mediated stabilization of KEAP1 in lipid-treated hepatocytes promotes oxidative stress and hepatic inflammation. (A-F) Subconfluent Huh7 Control or Huh7 TM4SF5-KO hepatocyte variants, either stably or transiently transfected with the indicated cDNAs, were analyzed for ROS production using flow cytometry following DCFDA staining. Cells received vehicle treatment (-), PA alone (B, D, and F), lipid mixture (LM, C), or DOX to induce Keap1 knockdown (shKEAP1 #2 or #3 , see Table , C and D). In some experiments, Huh7 cell variants were transfected with siNS (non-specific sequences as a control) or siNRF2 (targeting sequence #1 or #2, see Table ) for 24 h. Cells were then treated with DOX for shKEAP1 #2 induction for 24 h, followed by PA for 4 h prior to ROS analysis by flow cytometry (D). Cells were also exposed to H 2 O 2 at the indicated concentrations and durations before assessment of ROS levels (E). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns = non-significant, unpaired Student's t test or two-way ANOVA. Data are expressed as mean ± SEM. (G) Subconfluent SNU449 hepatocytes stably expressing empty vector (SNU449 EV ) or TM4SF5 (SNU449 TM4SF5 ) were harvested to perform qRT-PCR for the indicated molecules. (H) Subconfluent Huh7 Control cells were treated in the absence (-) or presence (+) of DOX to induce KEAP1 knockdown (shKEAP1 #2 ) for 24 h and then exposed to PA (100 μM) for 4 h before being collected for immunoblot analysis of the indicated molecules. (I) Murine AML12 cells stably transfected with EV or TM4SF5 were treated with 5% LM for 24 h, harvested, and subjected to qRT-PCR for the indicated molecules. Data shown are representative of three independent experiments. See also .

Article Snippet: The primary antibodies used included TM4SF5-EC2 and KEAP1 (sc-365626, Santa Cruz Biotech.).

Techniques: Control, Stable Transfection, Transfection, Flow Cytometry, Staining, Knockdown, Sequencing, Expressing, Plasmid Preparation, Quantitative RT-PCR, Western Blot

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: In vivo MASH-associated fibrosis model involves TM4SF5-dependent KEAP1 stabilization. (A-E) WT, Alb -TG Tm4sf5-Flag , and Tm4sf5 -/- KO C57BL/6 male mice (n=4~5, 8-week-old) were fed NCD or HFD without or with CCl 4 IP injection (HFDCCl 4 ) in the presence of vehicle DMSO or TSAHC treatment (IP injection, twice per week) for an additional 12 weeks until 20 weeks of age (A), after which they were euthanized for liver analysis using H&E, Masson's trichrome (M-T), Oil Red O (ORO), dihydroethidium (DHE) staining (B), immunohistochemistry (C), or immunoblotting (D and E). Scale bar: 100 μm. (F) Livers from animals fed only NCD or HFD for 12 weeks were processed to prepare whole tissue extracts for subsequent immunoblotting targeting the indicated molecules. (G) Liver tissues from animals treated as in (A) were analyzed by qRT-PCR for the target molecules. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns, non-significant, unpaired Student's t test or two-way ANOVA. Data are presented as mean ± SEM. Data shown are representative of three independent experiments. See also .

Article Snippet: The primary antibodies used included TM4SF5-EC2 and KEAP1 (sc-365626, Santa Cruz Biotech.).

Techniques: In Vivo, Injection, Staining, Immunohistochemistry, Western Blot, Quantitative RT-PCR

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: Both murine and human MASH models demonstrate involvement of Tm4sf5, Keap1, and CD36, likely independent of the DNA-binding ability of NRF2. (A-E) WT, Nrf2 mut , Alb -TG Tm4sf5 ×Nrf2 mut , Tm4sf5 -/- KO×Nrf2 mut male C57BL/6 mice (n = 4~5, 8-week-old) were fed either NCD or HFD (60% kcal fat) alongside intraperitoneal CCl 4 injection (5 mg/kg, twice per week) for 12 weeks, after which tissues were examined using H&E staining or immunohistochemistry for Keap1 (B), or subjected to immunoblotting for selected proteins (C-E). (F and G) Analyses of GSE135251 dataset from either healthy controls or MASLD patients with varying NAS (from 0 to 4) or GSE48452 dataset was conducted to compare gene expression levels among normal, NAFL, and MASH patient samples stratified by NAS. The TM4SF5 High or TM4SF5 Low classification was determined based on the top 50% or bottom 50% expression levels of TM4SF5 mRNA in the cohort. (H) Human liver biopsy samples from control individuals and MASLD patients with different NAS (from 0 to 4) were used to obtain whole tissue extracts for immunoblotting of designated proteins. Because biopsy samples were very small (~ 7 mm circular pillar with 1 mm diameter), sufficient protein extracts for all targets were not always available, resulting in variable patient numbers analyzed in each immunoblot. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns, non-significant, one-way ANOVA. Data are reported as mean ± SEM. Findings presented represent three independent experimental replicates. See also .

Article Snippet: The primary antibodies used included TM4SF5-EC2 and KEAP1 (sc-365626, Santa Cruz Biotech.).

Techniques: Binding Assay, Injection, Staining, Immunohistochemistry, Western Blot, Gene Expression, Expressing, Control

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: Keap1 downregulation reverses TM4SF5-driven, MASH-associated fibrosis upon MCD diet. (A-D) WT, Alb -TG Tm4sf5-Flag , and Tm4sf5 -/- KO C57BL/6 male mice (n=4~5, 8-week-old) were maintained on an MCD diet for 3.5 weeks (A) and received intravenous injections of either PBS or siKeap1 (0.02 mg/kg, twice a week) prior to sacrifice for subsequent hepatic analyses, including body and liver weight measurements (B), and evaluation through H&E, Masson's trichrome (M-T), Oil Red O (ORO), or dihydroethidium (DHE) staining, immunohistochemistry (C), or immunoblotting (D) for specified proteins. Scale bar: 100 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns, non-significant, one-way ANOVA. All data are expressed as mean ± SEM.

Article Snippet: The primary antibodies used included TM4SF5-EC2 and KEAP1 (sc-365626, Santa Cruz Biotech.).

Techniques: Staining, Immunohistochemistry, Western Blot

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: Schematic model illustrating the modulation of KEAP1 by TM4SF5 under basal and pathological conditions. (Left) In healthy livers, TM4SF5 expression is very low and can be further upregulated by a hepatic inflammatory environment, presumably during early homeostatic conditions with minimal features of fatty liver. In this context, KEAP1 maintains regulation of NRF2 for antioxidant gene induction. In livers exhibiting balanced lipid catabolism and anabolism, or in the presence of extracellular lipids that do not induce MASH [e.g., HFD , low levels of tetraspanin TM4SF5 associate with KEAP1 and CD36, promoting KEAP1 ubiquitination and degradation and consequently increasing CD36 levels, thereby supporting homeostatic lipid metabolism. (Right) In situations of greater extracellular lipid overload, which can trigger MASH, TM4SF5 enhances the production of inflammatory cytokines and chemokines and increases ROS accumulation. Concurrently, TM4SF5 expression is elevated due to inflammation and further stabilizes KEAP1, facilitating ROS sensing and the induction of lipogenic genes. This leads to dysregulated lipid uptake, likely due to decreased CD36 levels, and an increase in NAS (from F1 to F4). Accordingly, dysregulated lipid metabolism and the pro-inflammatory environment, including ROS accumulation in TM4SF5-positive hepatocytes or livers, can drive MASH through TM4SF5-mediated KEAP1 stabilization, presumably independent of NRF2 levels or transcriptional function.

Article Snippet: The primary antibodies used included TM4SF5-EC2 and KEAP1 (sc-365626, Santa Cruz Biotech.).

Techniques: Expressing, Ubiquitin Proteomics

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: Hepatocyte TM4SF5-mediated downregulation of protein KEAP1 via their binding is irrelevant to NRF2. Hepatocytes including TM4SF5-null cell lines [SNU449 lacking endogenously and Huh7 TM4SF5-KO via CRISPR/Cas9-mediated KO , TM4SF5-expressing cell lines (Hep3B and Huh7 Control expressing endogenously), or primary hepatocytes from C57BL/6 male mice (G) were cultured at subconfluent conditions. Cells were transfected either transiently or stably with the relevant cDNAs. Additionally, they were exposed to TSAHC (a TM4SF5-specific chalcone inhibitor) for the indicated durations at varying concentrations (I) or transfected with shRNA targeting either a control sequence (shNS) or shNRF2 (target sequences #1 or #2, see Table ). Whole cell extracts were prepared using lysis buffer containing Triton X-100, Brij58 (H), or Triton X-100 alone (A-G and I-L). Following normalization, proteins were processed for (immuno)precipitation using streptavidin-agarose beads, anti-NRF2 antibody, or anti-KEAP1 antibody, prior to immunoblot analysis for designated molecules. Data shown are representative of three independent experiments. See also and S2.

Article Snippet: Cell culture, transfection, and reagents: Human hepatocellular carcinoma cell lines without TM4SF5 expression (SNU449 and SNU761) and cell lines expressing TM4SF5 (Huh7, HepG2, and Hep3B) were acquired from the Korean Cell Line Bank (Seoul National University, Seoul, Korea).

Techniques: Binding Assay, CRISPR, Expressing, Control, Cell Culture, Transfection, Stable Transfection, shRNA, Sequencing, Lysis, Immunoprecipitation, Western Blot

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: Correlations among TM4SF5, KEAP1, and CD36 levels following extracellular lipid treatment. (A-H) Subconfluent Huh7 (A, B, D, E, F, G, H, and I) and SNU449 (B, C) hepatocytes, either left unmodified or subjected to knockout (KO), were stably or transiently transfected with the specified cDNAs. For KEAP1 suppression, cells were treated with doxycycline (DOX) to induce silencing (G). In selected experiments, a lipid mixture (LM, 5%) was administered for 24 h. Following treatment, whole cell lysates (WCL) were prepared using lysis buffer containing Triton X-100. Lysates were quantified and subjected to immunoblotting (A, B, C, and G) or (immuno)precipitation (D, E, F, and H) with streptavidin-agarose beads, anti-Flag, or anti-KEAP1 antibodies, followed by immunoblotting for the indicated proteins. (I) Huh7 TM4SF5-KO cells complemented with mCherry-TM4SF5 WT were replated onto collagen I-precoated slide glasses and treated with either vehicle or lipid mixture prior to immunostaining and imaging. Scale bar: 100 μm. Data represent three independent experiments. See also .

Article Snippet: Cell culture, transfection, and reagents: Human hepatocellular carcinoma cell lines without TM4SF5 expression (SNU449 and SNU761) and cell lines expressing TM4SF5 (Huh7, HepG2, and Hep3B) were acquired from the Korean Cell Line Bank (Seoul National University, Seoul, Korea).

Techniques: Knock-Out, Stable Transfection, Transfection, Lysis, Western Blot, Immunoprecipitation, Immunostaining, Imaging

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: TM4SF5-mediated stabilization of KEAP1 in lipid-treated hepatocytes promotes oxidative stress and hepatic inflammation. (A-F) Subconfluent Huh7 Control or Huh7 TM4SF5-KO hepatocyte variants, either stably or transiently transfected with the indicated cDNAs, were analyzed for ROS production using flow cytometry following DCFDA staining. Cells received vehicle treatment (-), PA alone (B, D, and F), lipid mixture (LM, C), or DOX to induce Keap1 knockdown (shKEAP1 #2 or #3 , see Table , C and D). In some experiments, Huh7 cell variants were transfected with siNS (non-specific sequences as a control) or siNRF2 (targeting sequence #1 or #2, see Table ) for 24 h. Cells were then treated with DOX for shKEAP1 #2 induction for 24 h, followed by PA for 4 h prior to ROS analysis by flow cytometry (D). Cells were also exposed to H 2 O 2 at the indicated concentrations and durations before assessment of ROS levels (E). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns = non-significant, unpaired Student's t test or two-way ANOVA. Data are expressed as mean ± SEM. (G) Subconfluent SNU449 hepatocytes stably expressing empty vector (SNU449 EV ) or TM4SF5 (SNU449 TM4SF5 ) were harvested to perform qRT-PCR for the indicated molecules. (H) Subconfluent Huh7 Control cells were treated in the absence (-) or presence (+) of DOX to induce KEAP1 knockdown (shKEAP1 #2 ) for 24 h and then exposed to PA (100 μM) for 4 h before being collected for immunoblot analysis of the indicated molecules. (I) Murine AML12 cells stably transfected with EV or TM4SF5 were treated with 5% LM for 24 h, harvested, and subjected to qRT-PCR for the indicated molecules. Data shown are representative of three independent experiments. See also .

Article Snippet: Cell culture, transfection, and reagents: Human hepatocellular carcinoma cell lines without TM4SF5 expression (SNU449 and SNU761) and cell lines expressing TM4SF5 (Huh7, HepG2, and Hep3B) were acquired from the Korean Cell Line Bank (Seoul National University, Seoul, Korea).

Techniques: Control, Stable Transfection, Transfection, Flow Cytometry, Staining, Knockdown, Sequencing, Expressing, Plasmid Preparation, Quantitative RT-PCR, Western Blot

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: In vivo MASH-associated fibrosis model involves TM4SF5-dependent KEAP1 stabilization. (A-E) WT, Alb -TG Tm4sf5-Flag , and Tm4sf5 -/- KO C57BL/6 male mice (n=4~5, 8-week-old) were fed NCD or HFD without or with CCl 4 IP injection (HFDCCl 4 ) in the presence of vehicle DMSO or TSAHC treatment (IP injection, twice per week) for an additional 12 weeks until 20 weeks of age (A), after which they were euthanized for liver analysis using H&E, Masson's trichrome (M-T), Oil Red O (ORO), dihydroethidium (DHE) staining (B), immunohistochemistry (C), or immunoblotting (D and E). Scale bar: 100 μm. (F) Livers from animals fed only NCD or HFD for 12 weeks were processed to prepare whole tissue extracts for subsequent immunoblotting targeting the indicated molecules. (G) Liver tissues from animals treated as in (A) were analyzed by qRT-PCR for the target molecules. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns, non-significant, unpaired Student's t test or two-way ANOVA. Data are presented as mean ± SEM. Data shown are representative of three independent experiments. See also .

Article Snippet: Cell culture, transfection, and reagents: Human hepatocellular carcinoma cell lines without TM4SF5 expression (SNU449 and SNU761) and cell lines expressing TM4SF5 (Huh7, HepG2, and Hep3B) were acquired from the Korean Cell Line Bank (Seoul National University, Seoul, Korea).

Techniques: In Vivo, Injection, Staining, Immunohistochemistry, Western Blot, Quantitative RT-PCR

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: Both murine and human MASH models demonstrate involvement of Tm4sf5, Keap1, and CD36, likely independent of the DNA-binding ability of NRF2. (A-E) WT, Nrf2 mut , Alb -TG Tm4sf5 ×Nrf2 mut , Tm4sf5 -/- KO×Nrf2 mut male C57BL/6 mice (n = 4~5, 8-week-old) were fed either NCD or HFD (60% kcal fat) alongside intraperitoneal CCl 4 injection (5 mg/kg, twice per week) for 12 weeks, after which tissues were examined using H&E staining or immunohistochemistry for Keap1 (B), or subjected to immunoblotting for selected proteins (C-E). (F and G) Analyses of GSE135251 dataset from either healthy controls or MASLD patients with varying NAS (from 0 to 4) or GSE48452 dataset was conducted to compare gene expression levels among normal, NAFL, and MASH patient samples stratified by NAS. The TM4SF5 High or TM4SF5 Low classification was determined based on the top 50% or bottom 50% expression levels of TM4SF5 mRNA in the cohort. (H) Human liver biopsy samples from control individuals and MASLD patients with different NAS (from 0 to 4) were used to obtain whole tissue extracts for immunoblotting of designated proteins. Because biopsy samples were very small (~ 7 mm circular pillar with 1 mm diameter), sufficient protein extracts for all targets were not always available, resulting in variable patient numbers analyzed in each immunoblot. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns, non-significant, one-way ANOVA. Data are reported as mean ± SEM. Findings presented represent three independent experimental replicates. See also .

Article Snippet: Cell culture, transfection, and reagents: Human hepatocellular carcinoma cell lines without TM4SF5 expression (SNU449 and SNU761) and cell lines expressing TM4SF5 (Huh7, HepG2, and Hep3B) were acquired from the Korean Cell Line Bank (Seoul National University, Seoul, Korea).

Techniques: Binding Assay, Injection, Staining, Immunohistochemistry, Western Blot, Gene Expression, Expressing, Control

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: Keap1 downregulation reverses TM4SF5-driven, MASH-associated fibrosis upon MCD diet. (A-D) WT, Alb -TG Tm4sf5-Flag , and Tm4sf5 -/- KO C57BL/6 male mice (n=4~5, 8-week-old) were maintained on an MCD diet for 3.5 weeks (A) and received intravenous injections of either PBS or siKeap1 (0.02 mg/kg, twice a week) prior to sacrifice for subsequent hepatic analyses, including body and liver weight measurements (B), and evaluation through H&E, Masson's trichrome (M-T), Oil Red O (ORO), or dihydroethidium (DHE) staining, immunohistochemistry (C), or immunoblotting (D) for specified proteins. Scale bar: 100 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns, non-significant, one-way ANOVA. All data are expressed as mean ± SEM.

Article Snippet: Cell culture, transfection, and reagents: Human hepatocellular carcinoma cell lines without TM4SF5 expression (SNU449 and SNU761) and cell lines expressing TM4SF5 (Huh7, HepG2, and Hep3B) were acquired from the Korean Cell Line Bank (Seoul National University, Seoul, Korea).

Techniques: Staining, Immunohistochemistry, Western Blot

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: Schematic model illustrating the modulation of KEAP1 by TM4SF5 under basal and pathological conditions. (Left) In healthy livers, TM4SF5 expression is very low and can be further upregulated by a hepatic inflammatory environment, presumably during early homeostatic conditions with minimal features of fatty liver. In this context, KEAP1 maintains regulation of NRF2 for antioxidant gene induction. In livers exhibiting balanced lipid catabolism and anabolism, or in the presence of extracellular lipids that do not induce MASH [e.g., HFD , low levels of tetraspanin TM4SF5 associate with KEAP1 and CD36, promoting KEAP1 ubiquitination and degradation and consequently increasing CD36 levels, thereby supporting homeostatic lipid metabolism. (Right) In situations of greater extracellular lipid overload, which can trigger MASH, TM4SF5 enhances the production of inflammatory cytokines and chemokines and increases ROS accumulation. Concurrently, TM4SF5 expression is elevated due to inflammation and further stabilizes KEAP1, facilitating ROS sensing and the induction of lipogenic genes. This leads to dysregulated lipid uptake, likely due to decreased CD36 levels, and an increase in NAS (from F1 to F4). Accordingly, dysregulated lipid metabolism and the pro-inflammatory environment, including ROS accumulation in TM4SF5-positive hepatocytes or livers, can drive MASH through TM4SF5-mediated KEAP1 stabilization, presumably independent of NRF2 levels or transcriptional function.

Article Snippet: Cell culture, transfection, and reagents: Human hepatocellular carcinoma cell lines without TM4SF5 expression (SNU449 and SNU761) and cell lines expressing TM4SF5 (Huh7, HepG2, and Hep3B) were acquired from the Korean Cell Line Bank (Seoul National University, Seoul, Korea).

Techniques: Expressing, Ubiquitin Proteomics

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: Hepatocyte TM4SF5-mediated downregulation of protein KEAP1 via their binding is irrelevant to NRF2. Hepatocytes including TM4SF5-null cell lines [SNU449 lacking endogenously and Huh7 TM4SF5-KO via CRISPR/Cas9-mediated KO , TM4SF5-expressing cell lines (Hep3B and Huh7 Control expressing endogenously), or primary hepatocytes from C57BL/6 male mice (G) were cultured at subconfluent conditions. Cells were transfected either transiently or stably with the relevant cDNAs. Additionally, they were exposed to TSAHC (a TM4SF5-specific chalcone inhibitor) for the indicated durations at varying concentrations (I) or transfected with shRNA targeting either a control sequence (shNS) or shNRF2 (target sequences #1 or #2, see Table ). Whole cell extracts were prepared using lysis buffer containing Triton X-100, Brij58 (H), or Triton X-100 alone (A-G and I-L). Following normalization, proteins were processed for (immuno)precipitation using streptavidin-agarose beads, anti-NRF2 antibody, or anti-KEAP1 antibody, prior to immunoblot analysis for designated molecules. Data shown are representative of three independent experiments. See also and S2.

Article Snippet: Cell culture, transfection, and reagents: Human hepatocellular carcinoma cell lines without TM4SF5 expression (SNU449 and SNU761) and cell lines expressing TM4SF5 (Huh7, HepG2, and Hep3B) were acquired from the Korean Cell Line Bank (Seoul National University, Seoul, Korea).

Techniques: Binding Assay, CRISPR, Expressing, Control, Cell Culture, Transfection, Stable Transfection, shRNA, Sequencing, Lysis, Immunoprecipitation, Western Blot

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: Correlations among TM4SF5, KEAP1, and CD36 levels following extracellular lipid treatment. (A-H) Subconfluent Huh7 (A, B, D, E, F, G, H, and I) and SNU449 (B, C) hepatocytes, either left unmodified or subjected to knockout (KO), were stably or transiently transfected with the specified cDNAs. For KEAP1 suppression, cells were treated with doxycycline (DOX) to induce silencing (G). In selected experiments, a lipid mixture (LM, 5%) was administered for 24 h. Following treatment, whole cell lysates (WCL) were prepared using lysis buffer containing Triton X-100. Lysates were quantified and subjected to immunoblotting (A, B, C, and G) or (immuno)precipitation (D, E, F, and H) with streptavidin-agarose beads, anti-Flag, or anti-KEAP1 antibodies, followed by immunoblotting for the indicated proteins. (I) Huh7 TM4SF5-KO cells complemented with mCherry-TM4SF5 WT were replated onto collagen I-precoated slide glasses and treated with either vehicle or lipid mixture prior to immunostaining and imaging. Scale bar: 100 μm. Data represent three independent experiments. See also .

Article Snippet: Cell culture, transfection, and reagents: Human hepatocellular carcinoma cell lines without TM4SF5 expression (SNU449 and SNU761) and cell lines expressing TM4SF5 (Huh7, HepG2, and Hep3B) were acquired from the Korean Cell Line Bank (Seoul National University, Seoul, Korea).

Techniques: Knock-Out, Stable Transfection, Transfection, Lysis, Western Blot, Immunoprecipitation, Immunostaining, Imaging

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: TM4SF5-mediated stabilization of KEAP1 in lipid-treated hepatocytes promotes oxidative stress and hepatic inflammation. (A-F) Subconfluent Huh7 Control or Huh7 TM4SF5-KO hepatocyte variants, either stably or transiently transfected with the indicated cDNAs, were analyzed for ROS production using flow cytometry following DCFDA staining. Cells received vehicle treatment (-), PA alone (B, D, and F), lipid mixture (LM, C), or DOX to induce Keap1 knockdown (shKEAP1 #2 or #3 , see Table , C and D). In some experiments, Huh7 cell variants were transfected with siNS (non-specific sequences as a control) or siNRF2 (targeting sequence #1 or #2, see Table ) for 24 h. Cells were then treated with DOX for shKEAP1 #2 induction for 24 h, followed by PA for 4 h prior to ROS analysis by flow cytometry (D). Cells were also exposed to H 2 O 2 at the indicated concentrations and durations before assessment of ROS levels (E). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns = non-significant, unpaired Student's t test or two-way ANOVA. Data are expressed as mean ± SEM. (G) Subconfluent SNU449 hepatocytes stably expressing empty vector (SNU449 EV ) or TM4SF5 (SNU449 TM4SF5 ) were harvested to perform qRT-PCR for the indicated molecules. (H) Subconfluent Huh7 Control cells were treated in the absence (-) or presence (+) of DOX to induce KEAP1 knockdown (shKEAP1 #2 ) for 24 h and then exposed to PA (100 μM) for 4 h before being collected for immunoblot analysis of the indicated molecules. (I) Murine AML12 cells stably transfected with EV or TM4SF5 were treated with 5% LM for 24 h, harvested, and subjected to qRT-PCR for the indicated molecules. Data shown are representative of three independent experiments. See also .

Article Snippet: Cell culture, transfection, and reagents: Human hepatocellular carcinoma cell lines without TM4SF5 expression (SNU449 and SNU761) and cell lines expressing TM4SF5 (Huh7, HepG2, and Hep3B) were acquired from the Korean Cell Line Bank (Seoul National University, Seoul, Korea).

Techniques: Control, Stable Transfection, Transfection, Flow Cytometry, Staining, Knockdown, Sequencing, Expressing, Plasmid Preparation, Quantitative RT-PCR, Western Blot

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: In vivo MASH-associated fibrosis model involves TM4SF5-dependent KEAP1 stabilization. (A-E) WT, Alb -TG Tm4sf5-Flag , and Tm4sf5 -/- KO C57BL/6 male mice (n=4~5, 8-week-old) were fed NCD or HFD without or with CCl 4 IP injection (HFDCCl 4 ) in the presence of vehicle DMSO or TSAHC treatment (IP injection, twice per week) for an additional 12 weeks until 20 weeks of age (A), after which they were euthanized for liver analysis using H&E, Masson's trichrome (M-T), Oil Red O (ORO), dihydroethidium (DHE) staining (B), immunohistochemistry (C), or immunoblotting (D and E). Scale bar: 100 μm. (F) Livers from animals fed only NCD or HFD for 12 weeks were processed to prepare whole tissue extracts for subsequent immunoblotting targeting the indicated molecules. (G) Liver tissues from animals treated as in (A) were analyzed by qRT-PCR for the target molecules. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns, non-significant, unpaired Student's t test or two-way ANOVA. Data are presented as mean ± SEM. Data shown are representative of three independent experiments. See also .

Article Snippet: Cell culture, transfection, and reagents: Human hepatocellular carcinoma cell lines without TM4SF5 expression (SNU449 and SNU761) and cell lines expressing TM4SF5 (Huh7, HepG2, and Hep3B) were acquired from the Korean Cell Line Bank (Seoul National University, Seoul, Korea).

Techniques: In Vivo, Injection, Staining, Immunohistochemistry, Western Blot, Quantitative RT-PCR

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: Both murine and human MASH models demonstrate involvement of Tm4sf5, Keap1, and CD36, likely independent of the DNA-binding ability of NRF2. (A-E) WT, Nrf2 mut , Alb -TG Tm4sf5 ×Nrf2 mut , Tm4sf5 -/- KO×Nrf2 mut male C57BL/6 mice (n = 4~5, 8-week-old) were fed either NCD or HFD (60% kcal fat) alongside intraperitoneal CCl 4 injection (5 mg/kg, twice per week) for 12 weeks, after which tissues were examined using H&E staining or immunohistochemistry for Keap1 (B), or subjected to immunoblotting for selected proteins (C-E). (F and G) Analyses of GSE135251 dataset from either healthy controls or MASLD patients with varying NAS (from 0 to 4) or GSE48452 dataset was conducted to compare gene expression levels among normal, NAFL, and MASH patient samples stratified by NAS. The TM4SF5 High or TM4SF5 Low classification was determined based on the top 50% or bottom 50% expression levels of TM4SF5 mRNA in the cohort. (H) Human liver biopsy samples from control individuals and MASLD patients with different NAS (from 0 to 4) were used to obtain whole tissue extracts for immunoblotting of designated proteins. Because biopsy samples were very small (~ 7 mm circular pillar with 1 mm diameter), sufficient protein extracts for all targets were not always available, resulting in variable patient numbers analyzed in each immunoblot. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns, non-significant, one-way ANOVA. Data are reported as mean ± SEM. Findings presented represent three independent experimental replicates. See also .

Article Snippet: Cell culture, transfection, and reagents: Human hepatocellular carcinoma cell lines without TM4SF5 expression (SNU449 and SNU761) and cell lines expressing TM4SF5 (Huh7, HepG2, and Hep3B) were acquired from the Korean Cell Line Bank (Seoul National University, Seoul, Korea).

Techniques: Binding Assay, Injection, Staining, Immunohistochemistry, Western Blot, Gene Expression, Expressing, Control

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: Keap1 downregulation reverses TM4SF5-driven, MASH-associated fibrosis upon MCD diet. (A-D) WT, Alb -TG Tm4sf5-Flag , and Tm4sf5 -/- KO C57BL/6 male mice (n=4~5, 8-week-old) were maintained on an MCD diet for 3.5 weeks (A) and received intravenous injections of either PBS or siKeap1 (0.02 mg/kg, twice a week) prior to sacrifice for subsequent hepatic analyses, including body and liver weight measurements (B), and evaluation through H&E, Masson's trichrome (M-T), Oil Red O (ORO), or dihydroethidium (DHE) staining, immunohistochemistry (C), or immunoblotting (D) for specified proteins. Scale bar: 100 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.0001; ns, non-significant, one-way ANOVA. All data are expressed as mean ± SEM.

Article Snippet: Cell culture, transfection, and reagents: Human hepatocellular carcinoma cell lines without TM4SF5 expression (SNU449 and SNU761) and cell lines expressing TM4SF5 (Huh7, HepG2, and Hep3B) were acquired from the Korean Cell Line Bank (Seoul National University, Seoul, Korea).

Techniques: Staining, Immunohistochemistry, Western Blot

Journal: International Journal of Biological Sciences

Article Title: TM4SF5-mediated KEAP1 Regulation in Hepatocytes Irrelevant to NRF2 Expression and Activity Promotes Oxidative Stress and Inflammation to Develop Metabolic Dysfunction-Associated Steatotic Liver Disease

doi: 10.7150/ijbs.126251

Figure Lengend Snippet: Schematic model illustrating the modulation of KEAP1 by TM4SF5 under basal and pathological conditions. (Left) In healthy livers, TM4SF5 expression is very low and can be further upregulated by a hepatic inflammatory environment, presumably during early homeostatic conditions with minimal features of fatty liver. In this context, KEAP1 maintains regulation of NRF2 for antioxidant gene induction. In livers exhibiting balanced lipid catabolism and anabolism, or in the presence of extracellular lipids that do not induce MASH [e.g., HFD , low levels of tetraspanin TM4SF5 associate with KEAP1 and CD36, promoting KEAP1 ubiquitination and degradation and consequently increasing CD36 levels, thereby supporting homeostatic lipid metabolism. (Right) In situations of greater extracellular lipid overload, which can trigger MASH, TM4SF5 enhances the production of inflammatory cytokines and chemokines and increases ROS accumulation. Concurrently, TM4SF5 expression is elevated due to inflammation and further stabilizes KEAP1, facilitating ROS sensing and the induction of lipogenic genes. This leads to dysregulated lipid uptake, likely due to decreased CD36 levels, and an increase in NAS (from F1 to F4). Accordingly, dysregulated lipid metabolism and the pro-inflammatory environment, including ROS accumulation in TM4SF5-positive hepatocytes or livers, can drive MASH through TM4SF5-mediated KEAP1 stabilization, presumably independent of NRF2 levels or transcriptional function.

Article Snippet: Cell culture, transfection, and reagents: Human hepatocellular carcinoma cell lines without TM4SF5 expression (SNU449 and SNU761) and cell lines expressing TM4SF5 (Huh7, HepG2, and Hep3B) were acquired from the Korean Cell Line Bank (Seoul National University, Seoul, Korea).

Techniques: Expressing, Ubiquitin Proteomics

Journal: BMB Reports

Article Title: Systemic TM4SF5 overexpression in Apc Min/+ mice promotes hepatic portal hypertension associated with fibrosis

doi: 10.5483/BMBRep.2022.55.12.104

Figure Lengend Snippet: Additional systemic overexpression of TM4SF5 in Apc Min/+ mice led to intramucosal adenocarcinomas in the intestines. We analyzed the intestines of Apc Min/+ ( n = 4) or Apc Min/+ :Tg TM4SF5 ( n = 6) mice at 26 weeks old using hematoxylin and eosin (H&E) staining. Tissues from two representative animals are shown separately in combined images (A, B). We quantified the pathological conditions (C).

Article Snippet: Antibodies that detect the human TM4SF5 EC2 (long extracellular loop) sequence or C-terminus sequence ( ) or the sequence ( 117 CLID NKWDYHFQETEGAYLRND138) in mouse TM4SF5 were custom designed (Pro-Sci, Poway, CA, USA).

Techniques: Over Expression, Staining

Journal: BMB Reports

Article Title: Systemic TM4SF5 overexpression in Apc Min/+ mice promotes hepatic portal hypertension associated with fibrosis

doi: 10.5483/BMBRep.2022.55.12.104

Figure Lengend Snippet: Additional systemic overexpression of TM4SF5 in Apc Min/+ mice increased β-catenin stabilization and transcriptional activity. (A) We analyzed the intestines of Apc Min/+ ( n = 4) or Apc Min/+ :Tg TM4SF5 ( n = 6) mice at 26 weeks old using immunohistochemistry (IHC) with mouse IgG or anti-β-catenin antibody. β-catenin immunostaining intensities were categorized as explained in the Materials and Methods section or quantitative comparison between conditions. (B) We analyzed HT29 cells via luciferase reporter assay following transfection with the β-catenin-responsive LEF/TCF-1 reporter pTOP-FLASH vector with shRNA plasmids (shControl or shTM4SF5) for 24 h; cells were treated with (+) or without (−) Wnt-3a for 12 h before the analysis. *, **, *** depict P < 0.05, 0.01, and 0.005, respectively. (C-E) HT29 (C, D) or HT116 (E) cells were independently transfected with control or Apc-full (C), shControl (shCon) or shTM4SF5 (D), or Mock-Flag or TM4SF5-Flag (E) plasmids for 24 h, and the cells were treated with recombinant Wnt-3a for 12 h, as explained in (B), prior to whole-cell extract preparation for standard Western blots for the indicated molecules. The data represent three independent experiments.

Article Snippet: Antibodies that detect the human TM4SF5 EC2 (long extracellular loop) sequence or C-terminus sequence ( ) or the sequence ( 117 CLID NKWDYHFQETEGAYLRND138) in mouse TM4SF5 were custom designed (Pro-Sci, Poway, CA, USA).

Techniques: Over Expression, Activity Assay, Immunohistochemistry, Immunostaining, Comparison, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, shRNA, Control, Recombinant, Western Blot

Journal: BMB Reports

Article Title: Systemic TM4SF5 overexpression in Apc Min/+ mice promotes hepatic portal hypertension associated with fibrosis

doi: 10.5483/BMBRep.2022.55.12.104

Figure Lengend Snippet: Apc Min/+ :Tg TM4SF5 mice showed sinusoidal dilatation, portal hypertension, and extracellular matrix (ECM) deposits in the liver. (A-C) We analyzed liver tissues from wild-type (WT, n = 4), Apc Min/+ ( n = 4), or Apc Min/+ :Tg TM4SF5 ( n = 6) mice at 26 weeks old using IHC with normal IgG, anti-TM4SF5, or anti-β-catenin antibody (A) and H&E staining or Masson’s trichrome staining (B). The liver tissues were also immunoblotted for the indicated molecules (C). (D) TM4SF5-null SNU449 or endogenously TM4SF5-expressing Hep3B hepatocytes were transfected with control, TM4SF5 WT, shControl, or shTM4SF5 #4 plasmids for 48 h, before whole cell lysate preparation for immunoblots for the indicated molecules. The data represent three independent experiments.

Article Snippet: Antibodies that detect the human TM4SF5 EC2 (long extracellular loop) sequence or C-terminus sequence ( ) or the sequence ( 117 CLID NKWDYHFQETEGAYLRND138) in mouse TM4SF5 were custom designed (Pro-Sci, Poway, CA, USA).

Techniques: Staining, Expressing, Transfection, Control, Western Blot

Journal: BMB Reports

Article Title: Systemic TM4SF5 overexpression in Apc Min/+ mice promotes hepatic portal hypertension associated with fibrosis

doi: 10.5483/BMBRep.2022.55.12.104

Figure Lengend Snippet: Tg TM4SF5 mice at 1.5 years old showed the phenotypes of portal hypertension associated with steatohepatitis and ECM deposits. We collected and analyzed liver tissues from 1.5-year-old C57BL/6 WT or Tg TM4SF5 male mice (n = 7) using H&E staining and Masson’s trichrome staining. Random representative images are shown. Image magnification is shown at 100× or 400×.

Article Snippet: Antibodies that detect the human TM4SF5 EC2 (long extracellular loop) sequence or C-terminus sequence ( ) or the sequence ( 117 CLID NKWDYHFQETEGAYLRND138) in mouse TM4SF5 were custom designed (Pro-Sci, Poway, CA, USA).

Techniques: Staining