murine aml12 hepatocyte cell line  (ATCC)

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: Communications Biology

Article Title: ABCC4 impairs the clearance of plasma LDL cholesterol through suppressing LDLR expression in the liver

doi: 10.1038/s42003-025-08818-x

Figure Lengend Snippet: a, b CRISPR/Cas9–mediated knockout of Abcc4 in AML12 cells ( a ) and LO2 cells ( b ) using two independent sgRNAs. Immunoblotting analysis of ABCC4 protein expression and Vinculin in control-KO cells and Abcc4 -KO cells (Rosa26 sgRNA, Abcc4 sgRNA #1, Abcc4 sgRNA #2). b Immunoblotting experiments of ABCC4 and Vinculin in control-KO cells and ABCC4-KO cells (AAVS1 sgRNA, ABCC4 sgRNA #1, #2). c Flow cytometry data showing that knockout of Abcc4 increased the abundance of LDLR on the AML12 cell surface (Representative data from n = 3 independent experiments with similar results). d Plot showing the relative Mean Fluorescence Intensity (MFI) of PE-LDLR from three independent experiments. e Immunoblotting analysis of plasma membrane fractions demonstrating that Abcc4 knockout dramatically increased the amount of LDLR on PM. f Quantification of band intensity of LDLR protein expression relative to Na/K-ATPase from three independent experiments. g Cultured AML12 cells were precooled to 4°C for 30 min and incubated with Dil-LDL for binding at 4°C. Then the cells were washed 3 times with ice-chilled PBS. The cells were substantially switched to 37 °C for uptake. h Representative immunofluorescence microscopy images showing that Abcc4 knockout in AML12 cells had potentiated influence on DiI-LDL binding (0 h) and uptake (1 h). Blue: DAPI; Red: Dil-LDL. Scale bar: 50 μm. i Dil-LDL uptake assay implying that LDL uptake was significantly promoted in Abcc4 -deficient cells by flow cytometry analysis (Representative data from n = 3 independent experiments with similar results). j The relative MFI of Dil-LDL quantification were from 3 independent experiments. k FC data showing that knockout of ABCC4 promotes the cell surface LDLR accessibility in LO2 cells (Representative data from n = 3 independent experiments with similar results). l The relative MFI of LDLR-PE quantification was from three independent experiments. m Immunoblotting analysis of LDLR quantification located on the plasma membrane fractions relative to Na/K-ATPase in LO2 cells. n Flow cytometry analysis showing that LDL uptake by LDLR was significantly promoted in LO2 cells lacking ABCC4 (Representative data from n = 3 independent experiments with similar results). o The relative MFI of Dil-LDL quantification were from three independent experiments. Statistical analysis was performed by a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test in ( d ), ( f ), ( j ), ( l ), ( o ). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ns: no significance. Data are the mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Murine hepatocyte cell line AML12 (Alpha Mouse Liver 12, ATCC CRL-2254) and human embryonic kidney (HEK) 293 T cell line (ATCC CRL-3216) were originally obtained from American Tissue Culture Collection Biobank (Manassas, VA, USA) and were incubated at 37°C with 5% CO2.

Techniques: CRISPR, Knock-Out, Western Blot, Expressing, Control, Flow Cytometry, Fluorescence, Clinical Proteomics, Membrane, Cell Culture, Incubation, Binding Assay, Immunofluorescence, Microscopy, Comparison

Journal: Communications Biology

Article Title: ABCC4 impairs the clearance of plasma LDL cholesterol through suppressing LDLR expression in the liver

doi: 10.1038/s42003-025-08818-x

Figure Lengend Snippet: a Flow cytometry data showing that ABCC4 inhibitor treatment potentiates surface LDLR availability (Representative data from n = 5 independent experiments with similar results). b Plot showing the relative Mean Fluorescence Intensity (MFI) of PE-LDLR from five independent experiments. c Immunoblotting experiments of the plasma membrane fractions demonstrating that ABCC4 inhibitor treatment up-regulates surface LDLR protein expression in AML12 cells. d Quantification of band intensity of surface LDLR protein expression relative to Na/K-ATPase in AML12 cells (Data from n = 3 independent experiments). e Wild-type male mice ( n = 6 per group) were injected intraperitoneally three times a week with a dose of Ceefourin-1 (10 mg/kg) or vehicle control (DMSO and corn oil) for 4 weeks under normal-chow diet (NCD) or high-fat diet (HFD) conditions. f Plot displaying changes of body weight in mice intraperitoneally with vehicle control and ABCC4 inhibitor Ceefourin-1 under a NCD or HFD condition. g Serum LDL-C level in WT mice treated as in ( e ). h Serum TC level in WT mice treated as in ( e ). i Serum TG level in WT mice treated as in ( e ). j Liver TC level in WT mice treated as in ( e ). k Liver TG level in WT mice treated as in ( e ). l Hematoxylin and eosin (H&E) and Oil Red O staining analysis revealing that ABCC4 inhibitor treatment improved lipid accumulation, especially under a HFD condition. Scale bar: 50 μm. m Immunoblotting data of LDLR protein expression from liver plasma membrane fractions in NDC-fed mice treated with Ceefourin-1 or vehicle. n Representative immunoblotting data of LDLR expression from liver plasma membrane fractions in HFD-fed mice treated with Ceefourin-1 or vehicle. o Quantification of band intensity of LDLR protein level relative to Na/K-ATPase in liver plasma membrane fractions from the mice treated as in ( e ). Statistical analysis was performed by an unpaired two-tailed Student’s t-test in ( b ), ( d ); a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test in ( g ), ( h ), ( i ), ( j ), ( k ), ( o ). * P ≤ 0.5, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ns: no significance. Data are the mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Murine hepatocyte cell line AML12 (Alpha Mouse Liver 12, ATCC CRL-2254) and human embryonic kidney (HEK) 293 T cell line (ATCC CRL-3216) were originally obtained from American Tissue Culture Collection Biobank (Manassas, VA, USA) and were incubated at 37°C with 5% CO2.

Techniques: Flow Cytometry, Fluorescence, Western Blot, Clinical Proteomics, Membrane, Expressing, Injection, Control, Staining, Two Tailed Test, Comparison

Journal: Communications Biology

Article Title: ABCC4 impairs the clearance of plasma LDL cholesterol through suppressing LDLR expression in the liver

doi: 10.1038/s42003-025-08818-x

Figure Lengend Snippet: a Principal component analysis showing distinct clustering of transcriptomes of the samples between Abcc4 -knockout (Abcc4 sgRNA) and control-knockout (Rosa26 sgRNA) AML12 cells. b Hierarchically clustered circos heatmap of top 43 differentially expressed genes (DEGs) between two groups ( P < 0.05; fold change>2.0). c KEGG pathway enrichment analysis of DEGs. d , e GSEA showing activated cAMP ( d ) and Rap1 ( e ) signaling pathways for DEGs between two groups. f Heatmap showing the enriched pathways related to lipid metabolism process, insulin secretion signaling, cAMP signaling, cGMP-PKG signaling, and Rap1 signaling for DEGs. g ELISA measurements showing increased intracellular and reduced extracellular cAMP levels in Abcc4 -deficient cells. h ELISA measurements showing increased intracellular and reduced extracellular cAMP levels in AML 12 cell treated with the ABCC4 inhibitor Ceefourin-1. i Immunoblotting experiments showing PCSK9 protein levels in Abcc4 -deficient cells. j Quantification of band intensity of PCSK9 protein expression relative to Vinculin in Abcc4 -deficient cells from three independent experiments. k Secreted PCSK9 levels in Abcc4 -deficient cells. l RT-qPCR results assessing Pcsk9 mRNA level in Abcc4 -deficient cells from three independent experiments. m Immunoblotting experiments showing PCSK9 protein level in AML12 cell treated with DMSO or Ceefourin-1. n Quantification of band intensity of PCSK9 protein level relative to Vinculin between two groups from three independent experiments. o Secreted PCSK9 levels in AML12 cells treated with DMSO or Ceefourin-1. p Pcsk9 relative mRNA expression level by RT-qPCR between two groups from three independent experiments. q Immunoblotting analysis of PCSK9 protein levels in Abcc4 -deficient cells treated with cycloheximide (cyclo: 4 μg/mL) for 30 min (+) and 90 min (++). r Flow cytometry analysis of Dil-LDL uptake assay (1 h) in Abcc4 -deficient cells treated with Vehicle or rhPCSK9 protein. The relative MFI of Dil-LDL quantification were from three independent experiments. s Immunoblotting analysis of LDLR expression from the plasma membrane fractions in Abcc4 -deficient cells. Statistical analysis was performed by a Welch ANOVA test followed by a post hoc analysis using the Tamhane T2 method in ( g ); an unpaired two-tailed Student’s t-test in ( h ), ( n ), ( o ), ( p ); a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test in ( j ), ( k ), ( l ), ( r ). * P ≤ 0.5, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ns: no significance. Data are the mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Murine hepatocyte cell line AML12 (Alpha Mouse Liver 12, ATCC CRL-2254) and human embryonic kidney (HEK) 293 T cell line (ATCC CRL-3216) were originally obtained from American Tissue Culture Collection Biobank (Manassas, VA, USA) and were incubated at 37°C with 5% CO2.

Techniques: Knock-Out, Control, Protein-Protein interactions, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Quantitative RT-PCR, Flow Cytometry, Clinical Proteomics, Membrane, Two Tailed Test, Comparison

Journal: Communications Biology

Article Title: ABCC4 impairs the clearance of plasma LDL cholesterol through suppressing LDLR expression in the liver

doi: 10.1038/s42003-025-08818-x

Figure Lengend Snippet: a Generation of Abcc4/Epac2 double-knockout (DKO) AML12 cells using two sgRNAs targeting Epac2 gene. Immunoblotting analysis of ABCC4, EPAC2 and Vinculin protein expression in AML12 cells (Rosa26 sgRNA, Abcc4 sgRNA #1, Abcc4 sgRNA #2, Abcc4/Epac2 sgRNA #1, Abcc4/Epac2 gRNA #2). b Immunoblotting analysis of LDLR protein expression from the plasma membrane fractions in AML12 cells (Rosa26 sgRNA, Abcc4 sgRNA #1, Abcc4 sgRNA #2, Abcc4/Epac2 sgRNA #1, Abcc4/Epac2 gRNA #2). c Flow cytometry analysis of Dil-LDL uptake assay (1 h) in AML12 cells (Rosa26 sgRNA, Abcc4 sgRNA, Abcc4/Epac2 sgRNA). d The relative MFI of Dil-LDL quantification were from 3 independent experiments. e Immunoblotting analysis of PCSK9 protein expression in AML12 cells (Rosa26 sgRNA, Abcc4 sgRNA #1, Abcc4 sgRNA #2, Abcc4/Epac2 sgRNA #1, Abcc4/Epac2 gRNA #2). f Secreted PCSK9 levels in AML12 cells (Rosa26 sgRNA, Abcc4 sgRNA, Abcc4/Epac2 sgRNA) from four independent experiments. g Relative Pcsk9 mRNA expression level in AML12 cells (Rosa26 sgRNA, Abcc4 sgRNA, Abcc4/Epac2 sgRNA) from four independent experiments. h Generation of Abcc4/Rap1a DKO AML12 cells using two independent sgRNAs targeting Rap1a gene. Immunoblotting analysis of ABCC4, RAP1A and Vinculin protein expression in AML12 cells (Rosa26 sgRNA, Abcc4 sgRNA #1, Abcc4 sgRNA #2, Abcc4/Rap1a sgRNA #1, Abcc4/Rap1a sgRNA #2). i Immunoblotting analysis of LDLR protein expression from the plasma membrane fractions in AML12 cells (Rosa26 sgRNA, Abcc4 sgRNA #1, Abcc4 sgRNA #2, Abcc4/Rap1a sgRNA #1, Abcc4/Rap1a sgRNA #2). j Flow cytometry analysis of Dil-LDL uptake assay (1 h) in AML12 cells (Rosa26 sgRNA, Abcc4 sgRNA, Abcc4/Rap1a sgRNA). k The relative MFI of Dil-LDL quantification were from 3 independent experiments. l Immunoblotting analysis of PCSK9 protein expression in AML12 cells (Rosa26 sgRNA, Abcc4 sgRNA #1, Abcc4 sgRNA #2, Abcc4/Rap1a sgRNA #1, Abcc4/Rap1a sgRNA #2). m Secreted PCSK9 levels in AML12 cells (Rosa26 sgRNA, Abcc4 sgRNA, Abcc4/Rap1a sgRNA) from four independent experiments . n Relative Pcsk9 mRNA expression level in AML12 cells (Rosa26 sgRNA, Abcc4 sgRNA, Abcc4/Rap1a sgRNA) from four independent experiments. Statistical analysis was performed by a Welch ANOVA test followed by a post hoc analysis using the Tamhane T2 method in ( d ), ( k ); a ordinary one-way ANOVA followed by Bonferroni’s multiple comparison test in ( f ), ( g ), ( m ), ( n ). * P ≤ 0.5, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ns: no significance. Data are the mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Murine hepatocyte cell line AML12 (Alpha Mouse Liver 12, ATCC CRL-2254) and human embryonic kidney (HEK) 293 T cell line (ATCC CRL-3216) were originally obtained from American Tissue Culture Collection Biobank (Manassas, VA, USA) and were incubated at 37°C with 5% CO2.

Techniques: Double Knockout, Western Blot, Expressing, Clinical Proteomics, Membrane, Flow Cytometry, Comparison