murine hepatocyte cell line aml12  (ATCC)


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    ATCC murine hepatocyte cell line aml12
    ( A ) Oil red O staining of liver sections of wild-type (WT) and Acod1 KO control and cecal slurry (CS) injected mice (5 μl/kg). Images are representative of five independent experiments. ( B ) Hepatic triglyceride content. n=7 mice per group. ( C ) Oleate-loaded primary hepatocytes (top panel) and <t>AML12</t> cells (bottom panel) were treated with vehicle (DMSO) or 4-octyl itaconate (4-OI) (250 µM) and stained with Nile Red (top panel) or BODIPY (bottom panel). Images arerepresentative of four independent experiments. Data are represented as mean ± SEM. *p<0.05, ***p<0.001. Scale bars are 50 μm.
    Murine Hepatocyte Cell Line Aml12, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Itaconate stabilizes CPT1a to enhance lipid utilization during inflammation"

    Article Title: Itaconate stabilizes CPT1a to enhance lipid utilization during inflammation

    Journal: eLife

    doi: 10.7554/eLife.92420

    ( A ) Oil red O staining of liver sections of wild-type (WT) and Acod1 KO control and cecal slurry (CS) injected mice (5 μl/kg). Images are representative of five independent experiments. ( B ) Hepatic triglyceride content. n=7 mice per group. ( C ) Oleate-loaded primary hepatocytes (top panel) and AML12 cells (bottom panel) were treated with vehicle (DMSO) or 4-octyl itaconate (4-OI) (250 µM) and stained with Nile Red (top panel) or BODIPY (bottom panel). Images arerepresentative of four independent experiments. Data are represented as mean ± SEM. *p<0.05, ***p<0.001. Scale bars are 50 μm.
    Figure Legend Snippet: ( A ) Oil red O staining of liver sections of wild-type (WT) and Acod1 KO control and cecal slurry (CS) injected mice (5 μl/kg). Images are representative of five independent experiments. ( B ) Hepatic triglyceride content. n=7 mice per group. ( C ) Oleate-loaded primary hepatocytes (top panel) and AML12 cells (bottom panel) were treated with vehicle (DMSO) or 4-octyl itaconate (4-OI) (250 µM) and stained with Nile Red (top panel) or BODIPY (bottom panel). Images arerepresentative of four independent experiments. Data are represented as mean ± SEM. *p<0.05, ***p<0.001. Scale bars are 50 μm.

    Techniques Used: Staining, Injection

    murine hepatocyte cell line aml12  (ATCC)


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    ATCC murine hepatocyte cell line aml12
    Murine Hepatocyte Cell Line Aml12, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    aml12 murine hepatocyte cell line  (ATCC)


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    ATCC aml12 murine hepatocyte cell line
    TGF-β1 inhibits cholesterol metabolism in hepatocytes. ( A ) Heatmap of the down-regulated genes involved in cholesterol metabolism upon 24-hour TGF-β1 treatment in <t>AML12</t> cells and MPHs. number of independent experiments [n] = 3. ( B ) The chord diagram illustrates the main biological processes of the decreased genes. ( C ) Networking interpretation of the down-regulated genes. ( D and E ) Signaling pathway analysis of the inhibited cholesterol metabolic genes. GO, Gene Ontology.
    Aml12 Murine Hepatocyte Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The Interplay of TGF-β1 and Cholesterol Orchestrating Hepatocyte Cell Fate, EMT, and Signals for HSC Activation"

    Article Title: The Interplay of TGF-β1 and Cholesterol Orchestrating Hepatocyte Cell Fate, EMT, and Signals for HSC Activation

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    doi: 10.1016/j.jcmgh.2023.12.012

    TGF-β1 inhibits cholesterol metabolism in hepatocytes. ( A ) Heatmap of the down-regulated genes involved in cholesterol metabolism upon 24-hour TGF-β1 treatment in AML12 cells and MPHs. number of independent experiments [n] = 3. ( B ) The chord diagram illustrates the main biological processes of the decreased genes. ( C ) Networking interpretation of the down-regulated genes. ( D and E ) Signaling pathway analysis of the inhibited cholesterol metabolic genes. GO, Gene Ontology.
    Figure Legend Snippet: TGF-β1 inhibits cholesterol metabolism in hepatocytes. ( A ) Heatmap of the down-regulated genes involved in cholesterol metabolism upon 24-hour TGF-β1 treatment in AML12 cells and MPHs. number of independent experiments [n] = 3. ( B ) The chord diagram illustrates the main biological processes of the decreased genes. ( C ) Networking interpretation of the down-regulated genes. ( D and E ) Signaling pathway analysis of the inhibited cholesterol metabolic genes. GO, Gene Ontology.

    Techniques Used:

    TGF-β1 inhibits cholesterol biosynthesis and accumulation in hepatocytes. ( A ) Relative mRNA levels of Hmgcr and Sqle were determined by RT-qPCR in AML12 cells treated with/without TGF-β1 for 24 hours. ( B ) Total cholesterol concentration was examined in AML12 cells treated (or not) with 2 or 5 ng/mL TGF-β1 for 72 hours. ( C ) Lipid droplet accumulation in AML12 cells was examined by BODIPY staining. Phalloidin was used for F-actin staining. ( D ) Quantification of F-actin staining. ( E ) Relative mRNA levels of TGF-β1 , and genes involved in cholesterol metabolism as shown in the figure were determined by RT-qPCR in liver tissue of mice treated with AAV8–control or AAV8–TGF-β1. ( F ) Protein expression of HMGCR in the liver tissue of control or AAV8–TGF-β1 mice was determined by Western blot. ( G ) Total cholesterol concentration was examined in the liver tissue of mice treated with AAV8–control or AAV8–TGF-β1. ( H ) Lipid droplet accumulation in liver tissue of mice treated with AAV8–control or AAV8–TGF-β1 was examined by BODIPY staining. ( I ) Quantification of BODIPY staining. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, and NS = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. For immunofluorescence, DRAQ5 was used to stain the nuclei. Scale bars : 25 μm. Images were chosen representatively from 3 independent experiments. Quantification of protein expression or staining was performed using ImageJ (National Institutes of Health). Chole., cholesterol; Con, control; conc., concentration; Fluor., fluorescence.
    Figure Legend Snippet: TGF-β1 inhibits cholesterol biosynthesis and accumulation in hepatocytes. ( A ) Relative mRNA levels of Hmgcr and Sqle were determined by RT-qPCR in AML12 cells treated with/without TGF-β1 for 24 hours. ( B ) Total cholesterol concentration was examined in AML12 cells treated (or not) with 2 or 5 ng/mL TGF-β1 for 72 hours. ( C ) Lipid droplet accumulation in AML12 cells was examined by BODIPY staining. Phalloidin was used for F-actin staining. ( D ) Quantification of F-actin staining. ( E ) Relative mRNA levels of TGF-β1 , and genes involved in cholesterol metabolism as shown in the figure were determined by RT-qPCR in liver tissue of mice treated with AAV8–control or AAV8–TGF-β1. ( F ) Protein expression of HMGCR in the liver tissue of control or AAV8–TGF-β1 mice was determined by Western blot. ( G ) Total cholesterol concentration was examined in the liver tissue of mice treated with AAV8–control or AAV8–TGF-β1. ( H ) Lipid droplet accumulation in liver tissue of mice treated with AAV8–control or AAV8–TGF-β1 was examined by BODIPY staining. ( I ) Quantification of BODIPY staining. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, and NS = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. For immunofluorescence, DRAQ5 was used to stain the nuclei. Scale bars : 25 μm. Images were chosen representatively from 3 independent experiments. Quantification of protein expression or staining was performed using ImageJ (National Institutes of Health). Chole., cholesterol; Con, control; conc., concentration; Fluor., fluorescence.

    Techniques Used: Quantitative RT-PCR, Concentration Assay, Staining, Expressing, Western Blot, Immunofluorescence, Fluorescence

    Alteration of the cholesterol level in AML12 cells affect different TGF-β1–mediated signaling to Smad2/3 and to AKT. ( A ) Total cholesterol concentration was examined in AML12 cells treated with 5 mmol/L cholesterol–MβCD complex (CE) or 50 μmol/L lovastatin and 50 μmol/L MVL (CD), respectively, for 14–16 hours. ( B ) AML12 cells treated for CE, CD, or untreated (control) then were stimulated with 2 ng/mL TGF-β1 for 2 hours. The levels of p-Smad2 and total Smad2 ( upper panels ) or p-Smad3 and total Smad3 ( lower panels ) were determined by Western blot analysis. ( C ) Cells treated to alter their cholesterol levels as described earlier were incubated with 2 ng/mL TGF-β1 for 30 minutes and subjected to Western blot of p-AKT and total AKT. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, NS = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Quantification of protein expression was performed using ImageJ (National Institutes of Health). Chole., cholesterol; p-AKT, phosphorylated-protein kinase B.
    Figure Legend Snippet: Alteration of the cholesterol level in AML12 cells affect different TGF-β1–mediated signaling to Smad2/3 and to AKT. ( A ) Total cholesterol concentration was examined in AML12 cells treated with 5 mmol/L cholesterol–MβCD complex (CE) or 50 μmol/L lovastatin and 50 μmol/L MVL (CD), respectively, for 14–16 hours. ( B ) AML12 cells treated for CE, CD, or untreated (control) then were stimulated with 2 ng/mL TGF-β1 for 2 hours. The levels of p-Smad2 and total Smad2 ( upper panels ) or p-Smad3 and total Smad3 ( lower panels ) were determined by Western blot analysis. ( C ) Cells treated to alter their cholesterol levels as described earlier were incubated with 2 ng/mL TGF-β1 for 30 minutes and subjected to Western blot of p-AKT and total AKT. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, NS = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Quantification of protein expression was performed using ImageJ (National Institutes of Health). Chole., cholesterol; p-AKT, phosphorylated-protein kinase B.

    Techniques Used: Concentration Assay, Western Blot, Incubation, Quantitative RT-PCR, Expressing

    CE inhibits TGF-β1–induced EMT and stress fiber formation whereas CD promotes these effects. ( A ) Brightfield images of the morphology of AML12 cells treated with CE/CD combined with/without 2 ng/mL TGF-β1 for 72 hours. Scale bars : 50 μm. ( B–D ) The expression and location of E-cadherin and fibronectin were determined by immunofluorescence in AML12 cells treated with CE/CD combined with/without 2 ng/mL TGF-β1 for 72 hours. ( E ) Relative mRNA levels of Twist , Fn1 , and Cdh1 , and ( F ) protein levels of E-cadherin were determined by RT-qPCR and Western blot, respectively, in AML12 cells with the conditioned treatment as shown in the figure. ( G ) Alexa Fluor 568 phalloidin staining showing actin polymerization in AML12 cells treated with CE/CD combined with/without 2 ng/mL TGF-β1 for 72 hours. ( H ) Quantification of phalloidin staining. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, NS = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. For immunofluorescence, DRAQ5 was used to stain the nuclei. Scale bars : 25 μm. Images were chosen representatively from 3 independent experiments. Quantification of protein expression or staining was performed using ImageJ (National Institutes of Health). E-cad, E-cadherin; Fluor., fluorescence.
    Figure Legend Snippet: CE inhibits TGF-β1–induced EMT and stress fiber formation whereas CD promotes these effects. ( A ) Brightfield images of the morphology of AML12 cells treated with CE/CD combined with/without 2 ng/mL TGF-β1 for 72 hours. Scale bars : 50 μm. ( B–D ) The expression and location of E-cadherin and fibronectin were determined by immunofluorescence in AML12 cells treated with CE/CD combined with/without 2 ng/mL TGF-β1 for 72 hours. ( E ) Relative mRNA levels of Twist , Fn1 , and Cdh1 , and ( F ) protein levels of E-cadherin were determined by RT-qPCR and Western blot, respectively, in AML12 cells with the conditioned treatment as shown in the figure. ( G ) Alexa Fluor 568 phalloidin staining showing actin polymerization in AML12 cells treated with CE/CD combined with/without 2 ng/mL TGF-β1 for 72 hours. ( H ) Quantification of phalloidin staining. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, NS = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. For immunofluorescence, DRAQ5 was used to stain the nuclei. Scale bars : 25 μm. Images were chosen representatively from 3 independent experiments. Quantification of protein expression or staining was performed using ImageJ (National Institutes of Health). E-cad, E-cadherin; Fluor., fluorescence.

    Techniques Used: Expressing, Immunofluorescence, Quantitative RT-PCR, Western Blot, Staining, Fluorescence

    CD-mediated EMT and actin polymerization depend on TGF-β1. ( A–D ) The expression and location of E-cadherin and fibronectin were determined by immunofluorescence in AML12 cells treated with CD combined with/without 10 μmol/L LY2157299 for 72 hours. ( E ) Relative mRNA levels of Twist , Fn1 , and Cdh1 , and ( F ) protein levels of E-cadherin were determined by RT-qPCR and Western blot, respectively, in AML12 cells with the conditioned treatment as shown in the figure. ( G ) Alexa Fluor 568 phalloidin staining showing actin polymerization in AML12 cells treated with CD combined with/without 10 μmol/L LY2157299 for 72 hours. ( H ) Quantification of phalloidin staining. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, ns = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. For immunofluorescence, DRAQ5 was used to stain the nuclei. Scale bars : 25 μm. Images were chosen representatively from 3 independent experiments. Quantification of protein expression or staining was performed using ImageJ (National Institutes of Health). Con, control; E-cad, E-cadherin; Fluor., fluorescence; LY, LY2157299.
    Figure Legend Snippet: CD-mediated EMT and actin polymerization depend on TGF-β1. ( A–D ) The expression and location of E-cadherin and fibronectin were determined by immunofluorescence in AML12 cells treated with CD combined with/without 10 μmol/L LY2157299 for 72 hours. ( E ) Relative mRNA levels of Twist , Fn1 , and Cdh1 , and ( F ) protein levels of E-cadherin were determined by RT-qPCR and Western blot, respectively, in AML12 cells with the conditioned treatment as shown in the figure. ( G ) Alexa Fluor 568 phalloidin staining showing actin polymerization in AML12 cells treated with CD combined with/without 10 μmol/L LY2157299 for 72 hours. ( H ) Quantification of phalloidin staining. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, ns = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. For immunofluorescence, DRAQ5 was used to stain the nuclei. Scale bars : 25 μm. Images were chosen representatively from 3 independent experiments. Quantification of protein expression or staining was performed using ImageJ (National Institutes of Health). Con, control; E-cad, E-cadherin; Fluor., fluorescence; LY, LY2157299.

    Techniques Used: Expressing, Immunofluorescence, Quantitative RT-PCR, Western Blot, Staining, Fluorescence

    CD induces cell apoptosis through the caspase 3/7 pathway. ( A ) The viability of AML12 cells treated with CE/CD ± TGF-β1 incubation for 72 hours was examined by MTT assay. ( B ) The confluency of AML12 cells subjected to the conditioned treatments indicated were analyzed by the IncuCyte machine. ( C ) Cell apoptosis was examined by IncuCyte Cytotox Red Dye in AML12 cells treated with/without CD ± TGF-β1 for 72 hours. Scale bars : 50 μm. ( D ) Cleaved caspase signals were tested by the caspase–Glo 3/7 assay kit in AML12 cells treated with/without CD ± TGF-β1 for 72 hours. ( E ) Protein levels of cleaved caspase 3/7 and caspase 3/7 were determined by Western blot in AML12 cells treated for CE/CD combined with/without 2 ng/mL TGF-β1 for 72 hours. ( F ) Cleaved caspase signals were tested by the caspase–Glo 3/7 assay kit in AML12 cells treated with/without CD ± TGF-β1 for 2 hours. ( G ) Protein levels of cleaved caspase 3/7, caspase 3/7, p-Smad2/3, and total Smad2/3 were determined by Western blot analysis in AML12 cells subjected (or not; FBS) to CE/CD, combined with/without 2 ng/mL TGF-β1 for 2 hours. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, ns = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Quantification of protein expression was performed using ImageJ (National Institutes of Health). Images were chosen representatively from 3 independent experiments. Con, control; RLU, relative light unit.
    Figure Legend Snippet: CD induces cell apoptosis through the caspase 3/7 pathway. ( A ) The viability of AML12 cells treated with CE/CD ± TGF-β1 incubation for 72 hours was examined by MTT assay. ( B ) The confluency of AML12 cells subjected to the conditioned treatments indicated were analyzed by the IncuCyte machine. ( C ) Cell apoptosis was examined by IncuCyte Cytotox Red Dye in AML12 cells treated with/without CD ± TGF-β1 for 72 hours. Scale bars : 50 μm. ( D ) Cleaved caspase signals were tested by the caspase–Glo 3/7 assay kit in AML12 cells treated with/without CD ± TGF-β1 for 72 hours. ( E ) Protein levels of cleaved caspase 3/7 and caspase 3/7 were determined by Western blot in AML12 cells treated for CE/CD combined with/without 2 ng/mL TGF-β1 for 72 hours. ( F ) Cleaved caspase signals were tested by the caspase–Glo 3/7 assay kit in AML12 cells treated with/without CD ± TGF-β1 for 2 hours. ( G ) Protein levels of cleaved caspase 3/7, caspase 3/7, p-Smad2/3, and total Smad2/3 were determined by Western blot analysis in AML12 cells subjected (or not; FBS) to CE/CD, combined with/without 2 ng/mL TGF-β1 for 2 hours. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, ns = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Quantification of protein expression was performed using ImageJ (National Institutes of Health). Images were chosen representatively from 3 independent experiments. Con, control; RLU, relative light unit.

    Techniques Used: Incubation, MTT Assay, Caspase-Glo Assay, Western Blot, Quantitative RT-PCR, Expressing

    CD promotes cell apoptosis through TGF-β1 signaling. ( A ) Brightfield images of AML12 cells treated (72 h) with CD alone, LY2157299 (10 μmol/L), or their combination. Untreated cells served as control. Scale bars : 50 μm. ( B ) The viability of AML12 cells treated for 72 hours with/without CD ± 10 μmol/L LY2157299 was examined by MTT assay. ( C ) Cleaved caspase signals were tested by the caspase–Glo 3/7 assay kit on AML12 cells subjected (or not; control) to CD treatment ± 10 μmol/L LY2157299 for 72 hours. ( D ) Western blot of cleaved and total caspase 3/7 in AML12 cells subjected to CD treatment with/without 10 μmol/L LY2157299 for 72 hours. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, NS = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. Quantification of protein expression was performed using ImageJ (National Institutes of Health). Images were chosen representatively from 3 independent experiments. Con, control; RLU, relative light unit.
    Figure Legend Snippet: CD promotes cell apoptosis through TGF-β1 signaling. ( A ) Brightfield images of AML12 cells treated (72 h) with CD alone, LY2157299 (10 μmol/L), or their combination. Untreated cells served as control. Scale bars : 50 μm. ( B ) The viability of AML12 cells treated for 72 hours with/without CD ± 10 μmol/L LY2157299 was examined by MTT assay. ( C ) Cleaved caspase signals were tested by the caspase–Glo 3/7 assay kit on AML12 cells subjected (or not; control) to CD treatment ± 10 μmol/L LY2157299 for 72 hours. ( D ) Western blot of cleaved and total caspase 3/7 in AML12 cells subjected to CD treatment with/without 10 μmol/L LY2157299 for 72 hours. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, NS = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. Quantification of protein expression was performed using ImageJ (National Institutes of Health). Images were chosen representatively from 3 independent experiments. Con, control; RLU, relative light unit.

    Techniques Used: MTT Assay, Caspase-Glo Assay, Western Blot, Quantitative RT-PCR, Expressing

    Conditioned media from CE- and CD-treated AML12 cells have opposite effects on HSC activation. ( A ) Active TGF-β1 concentration in the culture medium of LX-2 cells treated with conditioned supernatant of AML12 cells was examined by SEAP activity assay. ( B ) Relative mRNA levels of COL1A1 , COL3A1 , MMP2 , and MMP9 were determined by RT-qPCR in LX-2 cells treated with conditioned AML12 supernatants as shown in the figure. ( C ) Expression and location of TGF-β1 LAP-D (R58) were detected by immunofluorescent staining in LX-2 cells incubated with control or CD-treated supernatants of AML12 cells. ( D ) Quantification of TGF-β1 LAP-D (R58) staining. For RT-qPCR, human PPIA was used as the endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01. For immunofluorescence, DRAQ5 was used to stain the nuclei. Scale bars : 25 μm. Images were chosen representatively from 3 independent experiments. Quantification of staining was performed using ImageJ (National Institutes of Health). CD-SN, cholesterol depletion-treated cell supernatant; Con, control; Fluor., fluorescence.
    Figure Legend Snippet: Conditioned media from CE- and CD-treated AML12 cells have opposite effects on HSC activation. ( A ) Active TGF-β1 concentration in the culture medium of LX-2 cells treated with conditioned supernatant of AML12 cells was examined by SEAP activity assay. ( B ) Relative mRNA levels of COL1A1 , COL3A1 , MMP2 , and MMP9 were determined by RT-qPCR in LX-2 cells treated with conditioned AML12 supernatants as shown in the figure. ( C ) Expression and location of TGF-β1 LAP-D (R58) were detected by immunofluorescent staining in LX-2 cells incubated with control or CD-treated supernatants of AML12 cells. ( D ) Quantification of TGF-β1 LAP-D (R58) staining. For RT-qPCR, human PPIA was used as the endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01. For immunofluorescence, DRAQ5 was used to stain the nuclei. Scale bars : 25 μm. Images were chosen representatively from 3 independent experiments. Quantification of staining was performed using ImageJ (National Institutes of Health). CD-SN, cholesterol depletion-treated cell supernatant; Con, control; Fluor., fluorescence.

    Techniques Used: Activation Assay, Concentration Assay, Activity Assay, Quantitative RT-PCR, Expressing, Staining, Incubation, Immunofluorescence, Fluorescence

    aml12 murine hepatocyte cell line  (ATCC)


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    ATCC aml12 murine hepatocyte cell line
    Aml12 Murine Hepatocyte Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/aml12 murine hepatocyte cell line/product/ATCC
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    aml12 murine hepatocyte cell line - by Bioz Stars, 2024-04
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    murine hepatocyte cell line aml12  (ATCC)


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    ATCC murine hepatocyte cell line aml12
    The metabolic effects of TM4SF5‐enriched MLCSs on cellular respiratory reprogramming. (A) Primary hepatocytes isolated from WT or Tg Alb ‐Tm4sf5 C57BL/6N mice ( n = 4) were infected with HA‐OMP25 lentivirus. Mitochondria were pulled down using anti‐HA antibody beads for LC/MS proteomics analysis. (B) Huh7 cells with or without TM4SF5 suppression were glucose depleted and replete and processed for ETC complex‐activity assay. FCs in activity between glucose repletion and depletion were graphed as the mean ± SD. (C) SNU449 cells transfected with mCherry‐TM4SF5 and mito‐BFP were treated with 10 μmol/L TopFluor‐cholesterol for 1 h. Representative snapshots from confocal live imaging are presented. Quantitative analysis of the mitochondrial fission events was performed as explained in the Materials and Methods. (D) <t>AML12</t> cells transfected with the indicated constructs were treated without (‐) or with (+) 16 μmol/L cholesterol for 16 h before OCR measurements. AUCs for the OCR measurements were graphed. (E‐G) Primary hepatocytes from WT or Tm4sf5 −/‐ KO mice (E), WT mice preinjected with AAV8‐control or AAV8‐ Tbg ‐Tm4sf5 virus (F), or AML12 cells transfected with the indicated constructs (G) were processed for OCR measurements. (H) SNU449 cells stably infected with cDNA plasmids were glucose depleted and replete as indicated before harvests and immunoblots. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = non‐significant. Data were represented as mean ± SD. Data represent three independent experiments. See also Supplementary Figure . AAV8, adeno‐associated virus serotype 8; AUC, area under the curve; ETC, electron transport chain; FC, fold change; KO, knockout; LC/MS, Liquid chromatography / mass spectrometry, AML12, normal murine hepatocytes; MLCS, mitochondria‐lysosome contact site; OCR, oxygen consumption rate; SD, standard deviation; TM4SF5, transmembrane 4 L six family member 5; WT, wild‐type.
    Murine Hepatocyte Cell Line Aml12, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/murine hepatocyte cell line aml12/product/ATCC
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    murine hepatocyte cell line aml12 - by Bioz Stars, 2024-04
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    Images

    1) Product Images from "Glucose‐mediated mitochondrial reprogramming by cholesterol export at TM4SF5‐enriched mitochondria‐lysosome contact sites"

    Article Title: Glucose‐mediated mitochondrial reprogramming by cholesterol export at TM4SF5‐enriched mitochondria‐lysosome contact sites

    Journal: Cancer Communications

    doi: 10.1002/cac2.12510

    The metabolic effects of TM4SF5‐enriched MLCSs on cellular respiratory reprogramming. (A) Primary hepatocytes isolated from WT or Tg Alb ‐Tm4sf5 C57BL/6N mice ( n = 4) were infected with HA‐OMP25 lentivirus. Mitochondria were pulled down using anti‐HA antibody beads for LC/MS proteomics analysis. (B) Huh7 cells with or without TM4SF5 suppression were glucose depleted and replete and processed for ETC complex‐activity assay. FCs in activity between glucose repletion and depletion were graphed as the mean ± SD. (C) SNU449 cells transfected with mCherry‐TM4SF5 and mito‐BFP were treated with 10 μmol/L TopFluor‐cholesterol for 1 h. Representative snapshots from confocal live imaging are presented. Quantitative analysis of the mitochondrial fission events was performed as explained in the Materials and Methods. (D) AML12 cells transfected with the indicated constructs were treated without (‐) or with (+) 16 μmol/L cholesterol for 16 h before OCR measurements. AUCs for the OCR measurements were graphed. (E‐G) Primary hepatocytes from WT or Tm4sf5 −/‐ KO mice (E), WT mice preinjected with AAV8‐control or AAV8‐ Tbg ‐Tm4sf5 virus (F), or AML12 cells transfected with the indicated constructs (G) were processed for OCR measurements. (H) SNU449 cells stably infected with cDNA plasmids were glucose depleted and replete as indicated before harvests and immunoblots. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = non‐significant. Data were represented as mean ± SD. Data represent three independent experiments. See also Supplementary Figure . AAV8, adeno‐associated virus serotype 8; AUC, area under the curve; ETC, electron transport chain; FC, fold change; KO, knockout; LC/MS, Liquid chromatography / mass spectrometry, AML12, normal murine hepatocytes; MLCS, mitochondria‐lysosome contact site; OCR, oxygen consumption rate; SD, standard deviation; TM4SF5, transmembrane 4 L six family member 5; WT, wild‐type.
    Figure Legend Snippet: The metabolic effects of TM4SF5‐enriched MLCSs on cellular respiratory reprogramming. (A) Primary hepatocytes isolated from WT or Tg Alb ‐Tm4sf5 C57BL/6N mice ( n = 4) were infected with HA‐OMP25 lentivirus. Mitochondria were pulled down using anti‐HA antibody beads for LC/MS proteomics analysis. (B) Huh7 cells with or without TM4SF5 suppression were glucose depleted and replete and processed for ETC complex‐activity assay. FCs in activity between glucose repletion and depletion were graphed as the mean ± SD. (C) SNU449 cells transfected with mCherry‐TM4SF5 and mito‐BFP were treated with 10 μmol/L TopFluor‐cholesterol for 1 h. Representative snapshots from confocal live imaging are presented. Quantitative analysis of the mitochondrial fission events was performed as explained in the Materials and Methods. (D) AML12 cells transfected with the indicated constructs were treated without (‐) or with (+) 16 μmol/L cholesterol for 16 h before OCR measurements. AUCs for the OCR measurements were graphed. (E‐G) Primary hepatocytes from WT or Tm4sf5 −/‐ KO mice (E), WT mice preinjected with AAV8‐control or AAV8‐ Tbg ‐Tm4sf5 virus (F), or AML12 cells transfected with the indicated constructs (G) were processed for OCR measurements. (H) SNU449 cells stably infected with cDNA plasmids were glucose depleted and replete as indicated before harvests and immunoblots. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = non‐significant. Data were represented as mean ± SD. Data represent three independent experiments. See also Supplementary Figure . AAV8, adeno‐associated virus serotype 8; AUC, area under the curve; ETC, electron transport chain; FC, fold change; KO, knockout; LC/MS, Liquid chromatography / mass spectrometry, AML12, normal murine hepatocytes; MLCS, mitochondria‐lysosome contact site; OCR, oxygen consumption rate; SD, standard deviation; TM4SF5, transmembrane 4 L six family member 5; WT, wild‐type.

    Techniques Used: Isolation, Infection, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Transfection, Imaging, Construct, Virus, Stable Transfection, Western Blot, Knock-Out, Liquid Chromatography, Mass Spectrometry, Standard Deviation

    murine hepatocyte cell line aml12  (ATCC)


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    ATCC murine hepatocyte cell line aml12
    ( A ) Oil red O staining of liver sections of wild-type (WT) and Acod1 KO control and cecal slurry (CS) injected mice (5 μl/kg). Images are representative of five independent experiments. ( B ) Hepatic triglyceride content. n=7 mice per group. ( C ) Oleate-loaded primary hepatocytes (top panel) and <t>AML12</t> cells (bottom panel) were treated with vehicle (DMSO) or 4-octyl itaconate (4-OI) (250 µM) and stained with Nile Red (top panel) or BODIPY (bottom panel). Images arerepresentative of four independent experiments. Data are represented as mean ± SEM. *p<0.05, ***p<0.001. Scale bars are 50 μm.
    Murine Hepatocyte Cell Line Aml12, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC aml12 murine hepatocyte cell line
    TGF-β1 inhibits cholesterol metabolism in hepatocytes. ( A ) Heatmap of the down-regulated genes involved in cholesterol metabolism upon 24-hour TGF-β1 treatment in <t>AML12</t> cells and MPHs. number of independent experiments [n] = 3. ( B ) The chord diagram illustrates the main biological processes of the decreased genes. ( C ) Networking interpretation of the down-regulated genes. ( D and E ) Signaling pathway analysis of the inhibited cholesterol metabolic genes. GO, Gene Ontology.
    Aml12 Murine Hepatocyte Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC murine hepatocyte cell lines hcs aml12
    TGF-β1 inhibits cholesterol metabolism in hepatocytes. ( A ) Heatmap of the down-regulated genes involved in cholesterol metabolism upon 24-hour TGF-β1 treatment in <t>AML12</t> cells and MPHs. number of independent experiments [n] = 3. ( B ) The chord diagram illustrates the main biological processes of the decreased genes. ( C ) Networking interpretation of the down-regulated genes. ( D and E ) Signaling pathway analysis of the inhibited cholesterol metabolic genes. GO, Gene Ontology.
    Murine Hepatocyte Cell Lines Hcs Aml12, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC murine hepatic cell line aml12
    TGF-β1 inhibits cholesterol metabolism in hepatocytes. ( A ) Heatmap of the down-regulated genes involved in cholesterol metabolism upon 24-hour TGF-β1 treatment in <t>AML12</t> cells and MPHs. number of independent experiments [n] = 3. ( B ) The chord diagram illustrates the main biological processes of the decreased genes. ( C ) Networking interpretation of the down-regulated genes. ( D and E ) Signaling pathway analysis of the inhibited cholesterol metabolic genes. GO, Gene Ontology.
    Murine Hepatic Cell Line Aml12, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/murine hepatic cell line aml12/product/ATCC
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    ATCC aml12 murine hepatic cell line
    TGF-β1 inhibits cholesterol metabolism in hepatocytes. ( A ) Heatmap of the down-regulated genes involved in cholesterol metabolism upon 24-hour TGF-β1 treatment in <t>AML12</t> cells and MPHs. number of independent experiments [n] = 3. ( B ) The chord diagram illustrates the main biological processes of the decreased genes. ( C ) Networking interpretation of the down-regulated genes. ( D and E ) Signaling pathway analysis of the inhibited cholesterol metabolic genes. GO, Gene Ontology.
    Aml12 Murine Hepatic Cell Line, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/aml12 murine hepatic cell line/product/ATCC
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    Image Search Results


    ( A ) Oil red O staining of liver sections of wild-type (WT) and Acod1 KO control and cecal slurry (CS) injected mice (5 μl/kg). Images are representative of five independent experiments. ( B ) Hepatic triglyceride content. n=7 mice per group. ( C ) Oleate-loaded primary hepatocytes (top panel) and AML12 cells (bottom panel) were treated with vehicle (DMSO) or 4-octyl itaconate (4-OI) (250 µM) and stained with Nile Red (top panel) or BODIPY (bottom panel). Images arerepresentative of four independent experiments. Data are represented as mean ± SEM. *p<0.05, ***p<0.001. Scale bars are 50 μm.

    Journal: eLife

    Article Title: Itaconate stabilizes CPT1a to enhance lipid utilization during inflammation

    doi: 10.7554/eLife.92420

    Figure Lengend Snippet: ( A ) Oil red O staining of liver sections of wild-type (WT) and Acod1 KO control and cecal slurry (CS) injected mice (5 μl/kg). Images are representative of five independent experiments. ( B ) Hepatic triglyceride content. n=7 mice per group. ( C ) Oleate-loaded primary hepatocytes (top panel) and AML12 cells (bottom panel) were treated with vehicle (DMSO) or 4-octyl itaconate (4-OI) (250 µM) and stained with Nile Red (top panel) or BODIPY (bottom panel). Images arerepresentative of four independent experiments. Data are represented as mean ± SEM. *p<0.05, ***p<0.001. Scale bars are 50 μm.

    Article Snippet: Murine hepatocyte cell line AML12 (ATCC, #CRL-2254) were maintained on plastic cell culture plates in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F12 (DMEM/F-12) supplemented with 10% FBS, Gibco Insulin-Transferrin-Selenium supplement (Gibco), dexamethasone, and penicillin and streptomycin in a humidified incubator (37 °C, 5% CO 2 ).

    Techniques: Staining, Injection

    TGF-β1 inhibits cholesterol metabolism in hepatocytes. ( A ) Heatmap of the down-regulated genes involved in cholesterol metabolism upon 24-hour TGF-β1 treatment in AML12 cells and MPHs. number of independent experiments [n] = 3. ( B ) The chord diagram illustrates the main biological processes of the decreased genes. ( C ) Networking interpretation of the down-regulated genes. ( D and E ) Signaling pathway analysis of the inhibited cholesterol metabolic genes. GO, Gene Ontology.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: The Interplay of TGF-β1 and Cholesterol Orchestrating Hepatocyte Cell Fate, EMT, and Signals for HSC Activation

    doi: 10.1016/j.jcmgh.2023.12.012

    Figure Lengend Snippet: TGF-β1 inhibits cholesterol metabolism in hepatocytes. ( A ) Heatmap of the down-regulated genes involved in cholesterol metabolism upon 24-hour TGF-β1 treatment in AML12 cells and MPHs. number of independent experiments [n] = 3. ( B ) The chord diagram illustrates the main biological processes of the decreased genes. ( C ) Networking interpretation of the down-regulated genes. ( D and E ) Signaling pathway analysis of the inhibited cholesterol metabolic genes. GO, Gene Ontology.

    Article Snippet: The AML12 murine hepatocyte cell line (cat. CRL-2254; American Type Culture Collection, Manassas, VA) was established from hepatocytes of a mouse transgenic for human TGF-α.

    Techniques:

    TGF-β1 inhibits cholesterol biosynthesis and accumulation in hepatocytes. ( A ) Relative mRNA levels of Hmgcr and Sqle were determined by RT-qPCR in AML12 cells treated with/without TGF-β1 for 24 hours. ( B ) Total cholesterol concentration was examined in AML12 cells treated (or not) with 2 or 5 ng/mL TGF-β1 for 72 hours. ( C ) Lipid droplet accumulation in AML12 cells was examined by BODIPY staining. Phalloidin was used for F-actin staining. ( D ) Quantification of F-actin staining. ( E ) Relative mRNA levels of TGF-β1 , and genes involved in cholesterol metabolism as shown in the figure were determined by RT-qPCR in liver tissue of mice treated with AAV8–control or AAV8–TGF-β1. ( F ) Protein expression of HMGCR in the liver tissue of control or AAV8–TGF-β1 mice was determined by Western blot. ( G ) Total cholesterol concentration was examined in the liver tissue of mice treated with AAV8–control or AAV8–TGF-β1. ( H ) Lipid droplet accumulation in liver tissue of mice treated with AAV8–control or AAV8–TGF-β1 was examined by BODIPY staining. ( I ) Quantification of BODIPY staining. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, and NS = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. For immunofluorescence, DRAQ5 was used to stain the nuclei. Scale bars : 25 μm. Images were chosen representatively from 3 independent experiments. Quantification of protein expression or staining was performed using ImageJ (National Institutes of Health). Chole., cholesterol; Con, control; conc., concentration; Fluor., fluorescence.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: The Interplay of TGF-β1 and Cholesterol Orchestrating Hepatocyte Cell Fate, EMT, and Signals for HSC Activation

    doi: 10.1016/j.jcmgh.2023.12.012

    Figure Lengend Snippet: TGF-β1 inhibits cholesterol biosynthesis and accumulation in hepatocytes. ( A ) Relative mRNA levels of Hmgcr and Sqle were determined by RT-qPCR in AML12 cells treated with/without TGF-β1 for 24 hours. ( B ) Total cholesterol concentration was examined in AML12 cells treated (or not) with 2 or 5 ng/mL TGF-β1 for 72 hours. ( C ) Lipid droplet accumulation in AML12 cells was examined by BODIPY staining. Phalloidin was used for F-actin staining. ( D ) Quantification of F-actin staining. ( E ) Relative mRNA levels of TGF-β1 , and genes involved in cholesterol metabolism as shown in the figure were determined by RT-qPCR in liver tissue of mice treated with AAV8–control or AAV8–TGF-β1. ( F ) Protein expression of HMGCR in the liver tissue of control or AAV8–TGF-β1 mice was determined by Western blot. ( G ) Total cholesterol concentration was examined in the liver tissue of mice treated with AAV8–control or AAV8–TGF-β1. ( H ) Lipid droplet accumulation in liver tissue of mice treated with AAV8–control or AAV8–TGF-β1 was examined by BODIPY staining. ( I ) Quantification of BODIPY staining. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, and NS = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. For immunofluorescence, DRAQ5 was used to stain the nuclei. Scale bars : 25 μm. Images were chosen representatively from 3 independent experiments. Quantification of protein expression or staining was performed using ImageJ (National Institutes of Health). Chole., cholesterol; Con, control; conc., concentration; Fluor., fluorescence.

    Article Snippet: The AML12 murine hepatocyte cell line (cat. CRL-2254; American Type Culture Collection, Manassas, VA) was established from hepatocytes of a mouse transgenic for human TGF-α.

    Techniques: Quantitative RT-PCR, Concentration Assay, Staining, Expressing, Western Blot, Immunofluorescence, Fluorescence

    Alteration of the cholesterol level in AML12 cells affect different TGF-β1–mediated signaling to Smad2/3 and to AKT. ( A ) Total cholesterol concentration was examined in AML12 cells treated with 5 mmol/L cholesterol–MβCD complex (CE) or 50 μmol/L lovastatin and 50 μmol/L MVL (CD), respectively, for 14–16 hours. ( B ) AML12 cells treated for CE, CD, or untreated (control) then were stimulated with 2 ng/mL TGF-β1 for 2 hours. The levels of p-Smad2 and total Smad2 ( upper panels ) or p-Smad3 and total Smad3 ( lower panels ) were determined by Western blot analysis. ( C ) Cells treated to alter their cholesterol levels as described earlier were incubated with 2 ng/mL TGF-β1 for 30 minutes and subjected to Western blot of p-AKT and total AKT. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, NS = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Quantification of protein expression was performed using ImageJ (National Institutes of Health). Chole., cholesterol; p-AKT, phosphorylated-protein kinase B.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: The Interplay of TGF-β1 and Cholesterol Orchestrating Hepatocyte Cell Fate, EMT, and Signals for HSC Activation

    doi: 10.1016/j.jcmgh.2023.12.012

    Figure Lengend Snippet: Alteration of the cholesterol level in AML12 cells affect different TGF-β1–mediated signaling to Smad2/3 and to AKT. ( A ) Total cholesterol concentration was examined in AML12 cells treated with 5 mmol/L cholesterol–MβCD complex (CE) or 50 μmol/L lovastatin and 50 μmol/L MVL (CD), respectively, for 14–16 hours. ( B ) AML12 cells treated for CE, CD, or untreated (control) then were stimulated with 2 ng/mL TGF-β1 for 2 hours. The levels of p-Smad2 and total Smad2 ( upper panels ) or p-Smad3 and total Smad3 ( lower panels ) were determined by Western blot analysis. ( C ) Cells treated to alter their cholesterol levels as described earlier were incubated with 2 ng/mL TGF-β1 for 30 minutes and subjected to Western blot of p-AKT and total AKT. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, NS = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Quantification of protein expression was performed using ImageJ (National Institutes of Health). Chole., cholesterol; p-AKT, phosphorylated-protein kinase B.

    Article Snippet: The AML12 murine hepatocyte cell line (cat. CRL-2254; American Type Culture Collection, Manassas, VA) was established from hepatocytes of a mouse transgenic for human TGF-α.

    Techniques: Concentration Assay, Western Blot, Incubation, Quantitative RT-PCR, Expressing

    CE inhibits TGF-β1–induced EMT and stress fiber formation whereas CD promotes these effects. ( A ) Brightfield images of the morphology of AML12 cells treated with CE/CD combined with/without 2 ng/mL TGF-β1 for 72 hours. Scale bars : 50 μm. ( B–D ) The expression and location of E-cadherin and fibronectin were determined by immunofluorescence in AML12 cells treated with CE/CD combined with/without 2 ng/mL TGF-β1 for 72 hours. ( E ) Relative mRNA levels of Twist , Fn1 , and Cdh1 , and ( F ) protein levels of E-cadherin were determined by RT-qPCR and Western blot, respectively, in AML12 cells with the conditioned treatment as shown in the figure. ( G ) Alexa Fluor 568 phalloidin staining showing actin polymerization in AML12 cells treated with CE/CD combined with/without 2 ng/mL TGF-β1 for 72 hours. ( H ) Quantification of phalloidin staining. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, NS = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. For immunofluorescence, DRAQ5 was used to stain the nuclei. Scale bars : 25 μm. Images were chosen representatively from 3 independent experiments. Quantification of protein expression or staining was performed using ImageJ (National Institutes of Health). E-cad, E-cadherin; Fluor., fluorescence.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: The Interplay of TGF-β1 and Cholesterol Orchestrating Hepatocyte Cell Fate, EMT, and Signals for HSC Activation

    doi: 10.1016/j.jcmgh.2023.12.012

    Figure Lengend Snippet: CE inhibits TGF-β1–induced EMT and stress fiber formation whereas CD promotes these effects. ( A ) Brightfield images of the morphology of AML12 cells treated with CE/CD combined with/without 2 ng/mL TGF-β1 for 72 hours. Scale bars : 50 μm. ( B–D ) The expression and location of E-cadherin and fibronectin were determined by immunofluorescence in AML12 cells treated with CE/CD combined with/without 2 ng/mL TGF-β1 for 72 hours. ( E ) Relative mRNA levels of Twist , Fn1 , and Cdh1 , and ( F ) protein levels of E-cadherin were determined by RT-qPCR and Western blot, respectively, in AML12 cells with the conditioned treatment as shown in the figure. ( G ) Alexa Fluor 568 phalloidin staining showing actin polymerization in AML12 cells treated with CE/CD combined with/without 2 ng/mL TGF-β1 for 72 hours. ( H ) Quantification of phalloidin staining. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, NS = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. For immunofluorescence, DRAQ5 was used to stain the nuclei. Scale bars : 25 μm. Images were chosen representatively from 3 independent experiments. Quantification of protein expression or staining was performed using ImageJ (National Institutes of Health). E-cad, E-cadherin; Fluor., fluorescence.

    Article Snippet: The AML12 murine hepatocyte cell line (cat. CRL-2254; American Type Culture Collection, Manassas, VA) was established from hepatocytes of a mouse transgenic for human TGF-α.

    Techniques: Expressing, Immunofluorescence, Quantitative RT-PCR, Western Blot, Staining, Fluorescence

    CD-mediated EMT and actin polymerization depend on TGF-β1. ( A–D ) The expression and location of E-cadherin and fibronectin were determined by immunofluorescence in AML12 cells treated with CD combined with/without 10 μmol/L LY2157299 for 72 hours. ( E ) Relative mRNA levels of Twist , Fn1 , and Cdh1 , and ( F ) protein levels of E-cadherin were determined by RT-qPCR and Western blot, respectively, in AML12 cells with the conditioned treatment as shown in the figure. ( G ) Alexa Fluor 568 phalloidin staining showing actin polymerization in AML12 cells treated with CD combined with/without 10 μmol/L LY2157299 for 72 hours. ( H ) Quantification of phalloidin staining. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, ns = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. For immunofluorescence, DRAQ5 was used to stain the nuclei. Scale bars : 25 μm. Images were chosen representatively from 3 independent experiments. Quantification of protein expression or staining was performed using ImageJ (National Institutes of Health). Con, control; E-cad, E-cadherin; Fluor., fluorescence; LY, LY2157299.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: The Interplay of TGF-β1 and Cholesterol Orchestrating Hepatocyte Cell Fate, EMT, and Signals for HSC Activation

    doi: 10.1016/j.jcmgh.2023.12.012

    Figure Lengend Snippet: CD-mediated EMT and actin polymerization depend on TGF-β1. ( A–D ) The expression and location of E-cadherin and fibronectin were determined by immunofluorescence in AML12 cells treated with CD combined with/without 10 μmol/L LY2157299 for 72 hours. ( E ) Relative mRNA levels of Twist , Fn1 , and Cdh1 , and ( F ) protein levels of E-cadherin were determined by RT-qPCR and Western blot, respectively, in AML12 cells with the conditioned treatment as shown in the figure. ( G ) Alexa Fluor 568 phalloidin staining showing actin polymerization in AML12 cells treated with CD combined with/without 10 μmol/L LY2157299 for 72 hours. ( H ) Quantification of phalloidin staining. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, ns = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. For immunofluorescence, DRAQ5 was used to stain the nuclei. Scale bars : 25 μm. Images were chosen representatively from 3 independent experiments. Quantification of protein expression or staining was performed using ImageJ (National Institutes of Health). Con, control; E-cad, E-cadherin; Fluor., fluorescence; LY, LY2157299.

    Article Snippet: The AML12 murine hepatocyte cell line (cat. CRL-2254; American Type Culture Collection, Manassas, VA) was established from hepatocytes of a mouse transgenic for human TGF-α.

    Techniques: Expressing, Immunofluorescence, Quantitative RT-PCR, Western Blot, Staining, Fluorescence

    CD induces cell apoptosis through the caspase 3/7 pathway. ( A ) The viability of AML12 cells treated with CE/CD ± TGF-β1 incubation for 72 hours was examined by MTT assay. ( B ) The confluency of AML12 cells subjected to the conditioned treatments indicated were analyzed by the IncuCyte machine. ( C ) Cell apoptosis was examined by IncuCyte Cytotox Red Dye in AML12 cells treated with/without CD ± TGF-β1 for 72 hours. Scale bars : 50 μm. ( D ) Cleaved caspase signals were tested by the caspase–Glo 3/7 assay kit in AML12 cells treated with/without CD ± TGF-β1 for 72 hours. ( E ) Protein levels of cleaved caspase 3/7 and caspase 3/7 were determined by Western blot in AML12 cells treated for CE/CD combined with/without 2 ng/mL TGF-β1 for 72 hours. ( F ) Cleaved caspase signals were tested by the caspase–Glo 3/7 assay kit in AML12 cells treated with/without CD ± TGF-β1 for 2 hours. ( G ) Protein levels of cleaved caspase 3/7, caspase 3/7, p-Smad2/3, and total Smad2/3 were determined by Western blot analysis in AML12 cells subjected (or not; FBS) to CE/CD, combined with/without 2 ng/mL TGF-β1 for 2 hours. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, ns = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Quantification of protein expression was performed using ImageJ (National Institutes of Health). Images were chosen representatively from 3 independent experiments. Con, control; RLU, relative light unit.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: The Interplay of TGF-β1 and Cholesterol Orchestrating Hepatocyte Cell Fate, EMT, and Signals for HSC Activation

    doi: 10.1016/j.jcmgh.2023.12.012

    Figure Lengend Snippet: CD induces cell apoptosis through the caspase 3/7 pathway. ( A ) The viability of AML12 cells treated with CE/CD ± TGF-β1 incubation for 72 hours was examined by MTT assay. ( B ) The confluency of AML12 cells subjected to the conditioned treatments indicated were analyzed by the IncuCyte machine. ( C ) Cell apoptosis was examined by IncuCyte Cytotox Red Dye in AML12 cells treated with/without CD ± TGF-β1 for 72 hours. Scale bars : 50 μm. ( D ) Cleaved caspase signals were tested by the caspase–Glo 3/7 assay kit in AML12 cells treated with/without CD ± TGF-β1 for 72 hours. ( E ) Protein levels of cleaved caspase 3/7 and caspase 3/7 were determined by Western blot in AML12 cells treated for CE/CD combined with/without 2 ng/mL TGF-β1 for 72 hours. ( F ) Cleaved caspase signals were tested by the caspase–Glo 3/7 assay kit in AML12 cells treated with/without CD ± TGF-β1 for 2 hours. ( G ) Protein levels of cleaved caspase 3/7, caspase 3/7, p-Smad2/3, and total Smad2/3 were determined by Western blot analysis in AML12 cells subjected (or not; FBS) to CE/CD, combined with/without 2 ng/mL TGF-β1 for 2 hours. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, ns = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Quantification of protein expression was performed using ImageJ (National Institutes of Health). Images were chosen representatively from 3 independent experiments. Con, control; RLU, relative light unit.

    Article Snippet: The AML12 murine hepatocyte cell line (cat. CRL-2254; American Type Culture Collection, Manassas, VA) was established from hepatocytes of a mouse transgenic for human TGF-α.

    Techniques: Incubation, MTT Assay, Caspase-Glo Assay, Western Blot, Quantitative RT-PCR, Expressing

    CD promotes cell apoptosis through TGF-β1 signaling. ( A ) Brightfield images of AML12 cells treated (72 h) with CD alone, LY2157299 (10 μmol/L), or their combination. Untreated cells served as control. Scale bars : 50 μm. ( B ) The viability of AML12 cells treated for 72 hours with/without CD ± 10 μmol/L LY2157299 was examined by MTT assay. ( C ) Cleaved caspase signals were tested by the caspase–Glo 3/7 assay kit on AML12 cells subjected (or not; control) to CD treatment ± 10 μmol/L LY2157299 for 72 hours. ( D ) Western blot of cleaved and total caspase 3/7 in AML12 cells subjected to CD treatment with/without 10 μmol/L LY2157299 for 72 hours. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, NS = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. Quantification of protein expression was performed using ImageJ (National Institutes of Health). Images were chosen representatively from 3 independent experiments. Con, control; RLU, relative light unit.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: The Interplay of TGF-β1 and Cholesterol Orchestrating Hepatocyte Cell Fate, EMT, and Signals for HSC Activation

    doi: 10.1016/j.jcmgh.2023.12.012

    Figure Lengend Snippet: CD promotes cell apoptosis through TGF-β1 signaling. ( A ) Brightfield images of AML12 cells treated (72 h) with CD alone, LY2157299 (10 μmol/L), or their combination. Untreated cells served as control. Scale bars : 50 μm. ( B ) The viability of AML12 cells treated for 72 hours with/without CD ± 10 μmol/L LY2157299 was examined by MTT assay. ( C ) Cleaved caspase signals were tested by the caspase–Glo 3/7 assay kit on AML12 cells subjected (or not; control) to CD treatment ± 10 μmol/L LY2157299 for 72 hours. ( D ) Western blot of cleaved and total caspase 3/7 in AML12 cells subjected to CD treatment with/without 10 μmol/L LY2157299 for 72 hours. For RT-qPCR, mouse Ppia was used as endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01, NS = P > .05. For Western blot, glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. Quantification of protein expression was performed using ImageJ (National Institutes of Health). Images were chosen representatively from 3 independent experiments. Con, control; RLU, relative light unit.

    Article Snippet: The AML12 murine hepatocyte cell line (cat. CRL-2254; American Type Culture Collection, Manassas, VA) was established from hepatocytes of a mouse transgenic for human TGF-α.

    Techniques: MTT Assay, Caspase-Glo Assay, Western Blot, Quantitative RT-PCR, Expressing

    Conditioned media from CE- and CD-treated AML12 cells have opposite effects on HSC activation. ( A ) Active TGF-β1 concentration in the culture medium of LX-2 cells treated with conditioned supernatant of AML12 cells was examined by SEAP activity assay. ( B ) Relative mRNA levels of COL1A1 , COL3A1 , MMP2 , and MMP9 were determined by RT-qPCR in LX-2 cells treated with conditioned AML12 supernatants as shown in the figure. ( C ) Expression and location of TGF-β1 LAP-D (R58) were detected by immunofluorescent staining in LX-2 cells incubated with control or CD-treated supernatants of AML12 cells. ( D ) Quantification of TGF-β1 LAP-D (R58) staining. For RT-qPCR, human PPIA was used as the endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01. For immunofluorescence, DRAQ5 was used to stain the nuclei. Scale bars : 25 μm. Images were chosen representatively from 3 independent experiments. Quantification of staining was performed using ImageJ (National Institutes of Health). CD-SN, cholesterol depletion-treated cell supernatant; Con, control; Fluor., fluorescence.

    Journal: Cellular and Molecular Gastroenterology and Hepatology

    Article Title: The Interplay of TGF-β1 and Cholesterol Orchestrating Hepatocyte Cell Fate, EMT, and Signals for HSC Activation

    doi: 10.1016/j.jcmgh.2023.12.012

    Figure Lengend Snippet: Conditioned media from CE- and CD-treated AML12 cells have opposite effects on HSC activation. ( A ) Active TGF-β1 concentration in the culture medium of LX-2 cells treated with conditioned supernatant of AML12 cells was examined by SEAP activity assay. ( B ) Relative mRNA levels of COL1A1 , COL3A1 , MMP2 , and MMP9 were determined by RT-qPCR in LX-2 cells treated with conditioned AML12 supernatants as shown in the figure. ( C ) Expression and location of TGF-β1 LAP-D (R58) were detected by immunofluorescent staining in LX-2 cells incubated with control or CD-treated supernatants of AML12 cells. ( D ) Quantification of TGF-β1 LAP-D (R58) staining. For RT-qPCR, human PPIA was used as the endogenous control. Bars represent means ± SD (n = 3). ∗ P < .05, ∗∗ P < .01. For immunofluorescence, DRAQ5 was used to stain the nuclei. Scale bars : 25 μm. Images were chosen representatively from 3 independent experiments. Quantification of staining was performed using ImageJ (National Institutes of Health). CD-SN, cholesterol depletion-treated cell supernatant; Con, control; Fluor., fluorescence.

    Article Snippet: The AML12 murine hepatocyte cell line (cat. CRL-2254; American Type Culture Collection, Manassas, VA) was established from hepatocytes of a mouse transgenic for human TGF-α.

    Techniques: Activation Assay, Concentration Assay, Activity Assay, Quantitative RT-PCR, Expressing, Staining, Incubation, Immunofluorescence, Fluorescence