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 murine normal hepatocyte AML12 cell line, lacking TM4SF5, was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

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

Journal: Cells

Article Title: AUF1 Restrains Hepatocyte Senescence by Maintaining Mitochondrial Homeostasis in AML12 Hepatocyte Model

doi: 10.3390/cells15010048

Figure Lengend Snippet: Chronic oxidative stress induces senescence in AML12 hepatocytes and downregulates AUF1. A senescent hepatocyte model was established using AML12 cells as described in . Cell morphology ( A ) and number ( B ) were assessed on Days 0, 3, and 7. BrdU incorporation assay ( C ) and TUNEL assay ( D ) were used to determine cell proliferation and cell death. ( E ) SA β-gal staining. SA β-gal-positive cells were quantified by cell counting. ( F ) RT-qPCR. Gapdh mRNA was used for normalization. ( G ) Immunoblotting analysis. β-actin, a loading control. Pro, proliferating; Sen, senescent. Scale bar, 100 μm. Representative images from three independent biological replicates ( n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Mouse hepatocyte AML12 cells (American Type Culture Collection (ATCC), Manassas, VA, USA; CRL-2254) were purchased and cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F12 (DMEM/F12; Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% fetal bovine serum (FBS), 1% antibiotics, 1× Insulin-Transferrin-Selenium Pyruvate Supplement (ITSP; Welgene, Gyeongsan, Republic of Korea), and 100 nM dexamethasone (Sigma-Aldrich, Burlington, MA, USA) at 37 °C.

Techniques: BrdU Incorporation Assay, TUNEL Assay, Staining, Cell Counting, Quantitative RT-PCR, Western Blot, Control

Journal: Cells

Article Title: AUF1 Restrains Hepatocyte Senescence by Maintaining Mitochondrial Homeostasis in AML12 Hepatocyte Model

doi: 10.3390/cells15010048

Figure Lengend Snippet: Senescent AML12 hepatocytes show mitochondrial network elongation, MERCs reorganization, and bioenergetic impairment. ( A ) Mitochondrial morphology was analyzed by immunofluorescence microscopy using the NDUFV2 antibody. Mitochondrial number, area, perimeter, and branch length were quantified using ImageJ/Fiji software. Each dot represents a mitochondrial measurement from an individual cell. ( B ) TEM images. ( C ) Cellular ROS levels (CM-H 2 DCFDA staining). ( D ) Mitochondrial ROS levels (MitoSOX™ staining). ( E ) Mitochondrial ATP levels (Mitochondrial ToxGlo™). ( F ) Mitochondrial membrane potential (JC-1 staining). ( G , H ) MERCs were monitored using the split-GFP contact-site sensor SPLICS system ( G ), and mitochondrial Ca 2+ levels were determined using the mitochondria-targeted FRET probe 4mtD3cpv ( H ). Fluorescence of each probe was analyzed by ImageJ/Fiji software. Scale bar, 10 μm for ( A , H ), and 1 μm for ( B ). Representative images from three independent biological replicates ( n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Mouse hepatocyte AML12 cells (American Type Culture Collection (ATCC), Manassas, VA, USA; CRL-2254) were purchased and cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F12 (DMEM/F12; Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% fetal bovine serum (FBS), 1% antibiotics, 1× Insulin-Transferrin-Selenium Pyruvate Supplement (ITSP; Welgene, Gyeongsan, Republic of Korea), and 100 nM dexamethasone (Sigma-Aldrich, Burlington, MA, USA) at 37 °C.

Techniques: Immunofluorescence, Microscopy, Software, Staining, Membrane, Fluorescence

Journal: Cells

Article Title: AUF1 Restrains Hepatocyte Senescence by Maintaining Mitochondrial Homeostasis in AML12 Hepatocyte Model

doi: 10.3390/cells15010048

Figure Lengend Snippet: AUF1-KD promotes a senescent phenotype in AML12 hepatocytes. AML12 cells were transfected with siRNAs and cultured for 72 h. ( A ) AUF1 downregulation was confirmed by immunoblotting. ( B ) Cell morphology. ( C ) Cell number. ( D ) SA β-gal staining. SA β-gal-positive cells were quantified by cell counting. ( E ) RT-qPCR. Gapdh mRNA was used for normalization. ( F ) Immunoblot analysis. β-actin, a loading control. Scale bar, 100 μm. Representative images from three independent biological replicates ( n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Mouse hepatocyte AML12 cells (American Type Culture Collection (ATCC), Manassas, VA, USA; CRL-2254) were purchased and cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F12 (DMEM/F12; Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% fetal bovine serum (FBS), 1% antibiotics, 1× Insulin-Transferrin-Selenium Pyruvate Supplement (ITSP; Welgene, Gyeongsan, Republic of Korea), and 100 nM dexamethasone (Sigma-Aldrich, Burlington, MA, USA) at 37 °C.

Techniques: Transfection, Cell Culture, Western Blot, Staining, Cell Counting, Quantitative RT-PCR, Control

Journal: Cells

Article Title: AUF1 Restrains Hepatocyte Senescence by Maintaining Mitochondrial Homeostasis in AML12 Hepatocyte Model

doi: 10.3390/cells15010048

Figure Lengend Snippet: AUF1-KD results in senescence-like mitochondrial remodeling and reduces mitochondrial Ca 2+ uptake. AML12 cells were transfected with siRNAs and cultured for 72 h. ( A ) Mitochondrial morphology was analyzed by NDUFV2 immunofluorescence. Mitochondrial number, area, perimeter, and branch length of each image were analyzed using ImageJ/Fiji software. Each dot represents a mitochondrial measurement from an individual cell. ( B ) TEM images. ( C , D ) Cellular ROS levels (CM-H 2 DCFDA staining) and mitochondrial ROS levels (MitoSOX™ staining). ( E , F ) Mitochondrial ATP levels (Mitochondrial ToxGlo™) and mitochondrial membrane potential (JC-1 staining). ( G , H ) MERCs (SPLICS) ( G ) and mitochondrial Ca 2+ level (4mtD3cpv) ( H ). Fluorescence of each probe was analyzed by ImageJ/Fiji software. Scale bar, 10 μm for ( A , H ), and 1 μm for ( B ). Representative images from three independent biological replicates ( n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Mouse hepatocyte AML12 cells (American Type Culture Collection (ATCC), Manassas, VA, USA; CRL-2254) were purchased and cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F12 (DMEM/F12; Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% fetal bovine serum (FBS), 1% antibiotics, 1× Insulin-Transferrin-Selenium Pyruvate Supplement (ITSP; Welgene, Gyeongsan, Republic of Korea), and 100 nM dexamethasone (Sigma-Aldrich, Burlington, MA, USA) at 37 °C.

Techniques: Transfection, Cell Culture, Immunofluorescence, Software, Staining, Membrane, Fluorescence

Journal: Cells

Article Title: AUF1 Restrains Hepatocyte Senescence by Maintaining Mitochondrial Homeostasis in AML12 Hepatocyte Model

doi: 10.3390/cells15010048

Figure Lengend Snippet: AUF1-KD increases mitochondrial fusion factors and binds their 3′UTRs. ( A , B ) After siRNA transfection of AML12, levels of proteins and mRNAs were determined by immunoblotting ( A ) and RT-qPCR ( B ), respectively. Immunoblotting images were quantified using Image Lab software (Version 6.1). ( C ) RNP-IP followed by RT-qPCR. AUF1-containing RNP complexes were isolated, and relative mRNA enrichment was assessed by RT-qPCR compared with IgG control. ( D ) ( Upper ) Schematic representation of the 3′UTR of mouse Opa1 ( NM_001199177 ) and Mfn2 ( NM_001285920 ) mRNA. RNA probes (3U1 and 3U2) containing the GU/UG-rich region (yellow) and AU-rich region (cyan) of each 3′UTR were synthesized. ( Lower ) The binding between each probe and AUF1 was assessed biotin pulldown assay followed by immunoblotting. GAPDH 3U was used as a negative control for the biotin pulldown assay. β-actin, a loading control for immunoblotting. Gapdh mRNA, a reference gene for normalization. Representative images from independent biological replicates ( n = 3 for ( A – C ) and n = 2 for ( D )). * p < 0.05; ** p < 0.01.

Article Snippet: Mouse hepatocyte AML12 cells (American Type Culture Collection (ATCC), Manassas, VA, USA; CRL-2254) were purchased and cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F12 (DMEM/F12; Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% fetal bovine serum (FBS), 1% antibiotics, 1× Insulin-Transferrin-Selenium Pyruvate Supplement (ITSP; Welgene, Gyeongsan, Republic of Korea), and 100 nM dexamethasone (Sigma-Aldrich, Burlington, MA, USA) at 37 °C.

Techniques: Transfection, Western Blot, Quantitative RT-PCR, Software, Isolation, Control, Synthesized, Binding Assay, Negative Control

Journal: Cells

Article Title: AUF1 Restrains Hepatocyte Senescence by Maintaining Mitochondrial Homeostasis in AML12 Hepatocyte Model

doi: 10.3390/cells15010048

Figure Lengend Snippet: Senescence induction under AUF1-KD amplifies mitochondrial defects and senescence outputs. After siRNA transfection, senescence was induced in AML12 cells as described in Materials and Methods, and relative changes among proliferating control (Pro), senescent cells transfected with control siRNA (Sen-siCtrl), and senescent cells transfected with AUF1 siRNA (Sen-siAUF1) were analyzed. ( A ) Levels of Opa1 and Mfn2 mRNAs (RT-qPCR). ( B ) Levels of Opa1 and Mfn2 (immunoblotting). ( C ) Mitochondrial morphology analyzed by NDUFV2 immunofluorescence. Mitochondrial number, area, perimeter, and branch length were quantified using ImageJ/Fiji software. Each dot represents a mitochondrial measurement from an individual cell. ( D , E ) Cellular ROS levels (CM-H 2 DCFDA staining) and mitochondrial ROS levels (MitoSOX™ staining). ( F , G ) Mitochondrial ATP levels (Mitochondrial ToxGlo™) and mitochondrial membrane potential (JC-1 staining). ( H ) Levels of p16 , p21 , and p53 mRNAs (RT-qPCR) ( I ) Levels of p16 and p21 (immunoblotting) ( J ) SA β-gal staining. SA β-gal-positive cells were analyzed by cell counting. β-actin, a loading control for immunoblotting. Gapdh mRNA, a reference gene for normalization. Representative images from three independent biological replicates ( n = 3). Scale bar, 10 μm for ( C ), and 100 μm for ( J ). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Mouse hepatocyte AML12 cells (American Type Culture Collection (ATCC), Manassas, VA, USA; CRL-2254) were purchased and cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F12 (DMEM/F12; Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% fetal bovine serum (FBS), 1% antibiotics, 1× Insulin-Transferrin-Selenium Pyruvate Supplement (ITSP; Welgene, Gyeongsan, Republic of Korea), and 100 nM dexamethasone (Sigma-Aldrich, Burlington, MA, USA) at 37 °C.

Techniques: Transfection, Control, Quantitative RT-PCR, Western Blot, Immunofluorescence, Software, Staining, Membrane, Cell Counting

Journal: Cells

Article Title: AUF1 Restrains Hepatocyte Senescence by Maintaining Mitochondrial Homeostasis in AML12 Hepatocyte Model

doi: 10.3390/cells15010048

Figure Lengend Snippet: Ectopic expression of AUF1 preserves mitochondrial dynamics and attenuates senescence under pro-senescent stress. After adenoviral transduction, senescence was induced in AML12 cells, and relative changes among proliferating control (Pro), senescent cells transduced with control virus (Sen-Ad-Ctrl), and senescent cells transduced with AUF1 virus (Sen-Ad-AUF1) were analyzed. ( A ) Levels of Opa1 and Mfn2 mRNAs (RT-qPCR). ( B ) Levels of Opa1 and Mfn2 (immunoblotting). ( C ) Mitochondrial morphology was analyzed by NDUFV2 immunofluorescence. Mitochondrial number, area, perimeter, and branch length of each image were quantified using ImageJ/Fiji software. Each dot represents a mitochondrial measurement from an individual cell. ( D , E ) Cellular ROS levels (CM-H 2 DCFDA staining) and mitochondrial ROS levels (MitoSOX™ staining). ( F , G ) Mitochondrial ATP levels (Mitochondrial ToxGlo™) and mitochondrial membrane potential (JC-1 staining). ( H ) Levels of p16 , p21 , and p53 mRNAs (RT-qPCR) ( I ) Levels of p16 and p21 (immunoblotting) ( J ) SA β-gal staining. SA β-gal-positive cells were analyzed by cell counting. β-actin, a loading control for immunoblotting. Gapdh mRNA, a reference gene for normalization. Representative images from three independent biological replicates ( n = 3). Scale bar, 10 μm for ( C ), and 100 μm for ( J ). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Mouse hepatocyte AML12 cells (American Type Culture Collection (ATCC), Manassas, VA, USA; CRL-2254) were purchased and cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F12 (DMEM/F12; Capricorn Scientific, Ebsdorfergrund, Germany) supplemented with 10% fetal bovine serum (FBS), 1% antibiotics, 1× Insulin-Transferrin-Selenium Pyruvate Supplement (ITSP; Welgene, Gyeongsan, Republic of Korea), and 100 nM dexamethasone (Sigma-Aldrich, Burlington, MA, USA) at 37 °C.

Techniques: Expressing, Transduction, Control, Virus, Quantitative RT-PCR, Western Blot, Immunofluorescence, Software, Staining, Membrane, Cell Counting