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JCRB Cell Bank human hepatoma cell line huh-7 jcrb0403
Human Hepatoma Cell Line Huh 7 Jcrb0403, supplied by JCRB Cell Bank, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews

human hepatoma cell line huh-7 jcrb0403 - by Bioz Stars, 2026-10

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Article Title: CCIVR2 facilitates comprehensive identification of both overlapping and non-overlapping antisense transcripts within specified regions
Article Snippet: Human hepatoma cell line Huh-7 (JCRB0403) was purchased from JCRB Cell Bank (National Institute of Biomedical Innovation, Osaka, Japan), grown in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1 × penicillin/streptomycin (Meiji Seika Pharma Co., Ltd., Tokyo, Japan) at 37 °C under an atmosphere containing 5% CO 2 .

Article Title: CCIVR2 facilitates comprehensive identification of both overlapping and non-overlapping antisense transcripts within specified regions.
Article Snippet: Human hepatoma cell line Huh-7 (JCRB0403) was purchased from JCRB Cell Bank (National Institute of Biomedical Innovation, Osaka, Japan), grown in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1 × penicillin/streptomycin (Meiji Seika Pharma Co., Ltd., Tokyo, Japan) at 37 °C under an atmosphere containing 5% CO2.

Article Title: CCIVR facilitates comprehensive identification of cis-natural antisense transcripts with their structural characteristics and expression profiles.
Article Snippet: Human hepatoma cell line Huh-7 (JCRB0403) was purchased from JCRB Cell Bank (National Institute of Biomedical Innovation, Osaka, Japan), grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich) and 1 × penicillin/streptomycin (Meiji Seika Pharma Co., Ltd., Tokyo, Japan) at 37 °C under an atmosphere containing 5% CO2.

Article Title: CCIVR facilitates comprehensive identification of cis-natural antisense transcripts with their structural characteristics and expression profiles
Article Snippet: Human hepatoma cell line Huh-7 (JCRB0403) was purchased from JCRB Cell Bank (National Institute of Biomedical Innovation, Osaka, Japan), grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich) and 1 × penicillin/streptomycin (Meiji Seika Pharma Co., Ltd., Tokyo, Japan) at 37 °C under an atmosphere containing 5% CO 2 .

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Effect of kinase inhibitors on NTCP-mediated transport of taurocholate (TCA). (A) Accumulation of [ 3 H]-TCA (2 μM; 200 nCi/mL) in extracellular fluid buffer (ECF) with Na + over 10 min was measured in Mock-GFP- and NTCP-GFP-expressing HuH-7 cells in the presence of 0.1% DMSO control or various kinase inhibitors (10 µM). NTCP-GFP-expressing HuH-7 cells were pre-incubated for 15 min with 0.1% DMSO control (DMSO Ctrl) or kinase inhibitors (10 µM) in cell culture medium prior to initiation of the accumulation phase. Data were plotted as mean ± SD ( n = 3) and statistically significant differences compared to DMSO Ctrl were measured using an ordinary one-way ANOVA with Dunnett’s multiple comparison test (*, p < 0.05, **, p < 0.0001). (B) Mock-GFP or NTCP-GFP were induced in Flp-In ™ T-REx ™ 293 cells with 1 μg/mL doxycycline for three days and NTCP-GFP-expressing Flp-In ™ T-REx ™ 293 cells were pre-incubated with 0.1% DMSO control or 10 µM everolimus for 15 min in cell culture medium prior to initiation of the accumulation phase. Left panel: Accumulation of [ 3 H]-TCA (2 μM; 200 nCi/mL) in ECF without Na + (Chol + ) or with Na + over 10 min was measured in Mock-GFP- and NTCP-GFP-expressing Flp-In ™ T-REx ™ 293 cells in the presence of 0.1% DMSO control or 10 µM everolimus. Data were plotted as mean ± SEM ( n = 3, in triplicate) Right panel: Na + -dependent [ 3 H]-TCA accumulation was calculated based on the data in the left panel by subtracting [ 3 H]-TCA accumulation in Chol + buffer from [ 3 H]-TCA accumulation in Na + buffer. Data were plotted as mean ± SEM ( n = 3, in triplicate). Statistically significant differences compared to DMSO Ctrl were measured using an ordinary two-way ANOVA with Dunnett’s multiple comparison test (**, p < 0.0001; left panel) or an unpaired two-tailed t -test (*, p < 0.01; right panel).

Journal: Frontiers in Pharmacology

Article Title: Effect of mTOR inhibitors on sodium taurocholate cotransporting polypeptide (NTCP) function in vitro

doi: 10.3389/fphar.2023.1147495

Figure Lengend Snippet: Effect of kinase inhibitors on NTCP-mediated transport of taurocholate (TCA). (A) Accumulation of [ 3 H]-TCA (2 μM; 200 nCi/mL) in extracellular fluid buffer (ECF) with Na + over 10 min was measured in Mock-GFP- and NTCP-GFP-expressing HuH-7 cells in the presence of 0.1% DMSO control or various kinase inhibitors (10 µM). NTCP-GFP-expressing HuH-7 cells were pre-incubated for 15 min with 0.1% DMSO control (DMSO Ctrl) or kinase inhibitors (10 µM) in cell culture medium prior to initiation of the accumulation phase. Data were plotted as mean ± SD ( n = 3) and statistically significant differences compared to DMSO Ctrl were measured using an ordinary one-way ANOVA with Dunnett’s multiple comparison test (*, p < 0.05, **, p < 0.0001). (B) Mock-GFP or NTCP-GFP were induced in Flp-In ™ T-REx ™ 293 cells with 1 μg/mL doxycycline for three days and NTCP-GFP-expressing Flp-In ™ T-REx ™ 293 cells were pre-incubated with 0.1% DMSO control or 10 µM everolimus for 15 min in cell culture medium prior to initiation of the accumulation phase. Left panel: Accumulation of [ 3 H]-TCA (2 μM; 200 nCi/mL) in ECF without Na + (Chol + ) or with Na + over 10 min was measured in Mock-GFP- and NTCP-GFP-expressing Flp-In ™ T-REx ™ 293 cells in the presence of 0.1% DMSO control or 10 µM everolimus. Data were plotted as mean ± SEM ( n = 3, in triplicate) Right panel: Na + -dependent [ 3 H]-TCA accumulation was calculated based on the data in the left panel by subtracting [ 3 H]-TCA accumulation in Chol + buffer from [ 3 H]-TCA accumulation in Na + buffer. Data were plotted as mean ± SEM ( n = 3, in triplicate). Statistically significant differences compared to DMSO Ctrl were measured using an ordinary two-way ANOVA with Dunnett’s multiple comparison test (**, p < 0.0001; left panel) or an unpaired two-tailed t -test (*, p < 0.01; right panel).

Article Snippet: The human hepatoma HuH-7 cell line (JCRB0403) was purchased from Sekisui Xenotech (Kansas City, KS).

Techniques: Expressing, Incubation, Cell Culture, Two Tailed Test

Concentration-dependent inhibition of NTCP-mediated taurocholate (TCA) transport by everolimus. Accumulation of [ 3 H]-TCA (2 μM; 200 nCi/mL) in extracellular fluid buffer (ECF) with Na + over 10 min in the absence (DMSO Ctrl; 0.1%) and presence of everolimus (0.1–100 µM) was measured in (A) undifferentiated HuH-7 cells (HuH-7 Ctrl) and differentiated HuH-7 cells. Data were plotted as mean ± SD ( n = 3). Statistically significant differences compared to DMSO Ctrl were measured using an ordinary two-way ANOVA with Dunnett’s multiple comparison test (*, p < 0.05, **, p < 0.0001). (B) Left panel: Na + -dependent [ 3 H]-TCA accumulation was calculated by subtracting [ 3 H]-TCA accumulation in Chol + buffer from [ 3 H]-TCA accumulation in Na + buffer. Right panel: Relative Na + -dependent [ 3 H]-TCA transport was plotted with respect to everolimus concentration as a non-linear regression to calculate best fit IC 50 of 8.0 ± 1.3 µM. (C) Left panel: Mock-GFP or NTCP-GFP were induced in Flp-In ™ T-REx ™ 293 cells with 1 μg/mL doxycycline for three days. Accumulation of [ 3 H]-TCA (2 μM; 200 nCi/mL) in ECF with Na + over 10 min in the absence (DMSO Ctrl; 0.1%) and presence of everolimus (0.1–100 µM) was measured in stably transfected Mock-GFP and NTCP-GFP-expressing Flp-In ™ T-REx ™ 293 cells. Right panel: Relative [ 3 H]-TCA transport was plotted with respect to everolimus concentration as a non-linear regression to calculate best fit IC 50 of 6.7 ± 1.3 µM. All data were plotted as mean ± SD ( n = 3). Error bars are not visible when the SD is smaller than the data point. (B) and (C) Left panels: Statistically significant differences compared to DMSO Ctrl were measured using an ordinary one-way ANOVA with Dunnett's multiple comparison test (*, p < 0.05, **, p < 0.0001).

Journal: Frontiers in Pharmacology

Article Title: Effect of mTOR inhibitors on sodium taurocholate cotransporting polypeptide (NTCP) function in vitro

doi: 10.3389/fphar.2023.1147495

Figure Lengend Snippet: Concentration-dependent inhibition of NTCP-mediated taurocholate (TCA) transport by everolimus. Accumulation of [ 3 H]-TCA (2 μM; 200 nCi/mL) in extracellular fluid buffer (ECF) with Na + over 10 min in the absence (DMSO Ctrl; 0.1%) and presence of everolimus (0.1–100 µM) was measured in (A) undifferentiated HuH-7 cells (HuH-7 Ctrl) and differentiated HuH-7 cells. Data were plotted as mean ± SD ( n = 3). Statistically significant differences compared to DMSO Ctrl were measured using an ordinary two-way ANOVA with Dunnett’s multiple comparison test (*, p < 0.05, **, p < 0.0001). (B) Left panel: Na + -dependent [ 3 H]-TCA accumulation was calculated by subtracting [ 3 H]-TCA accumulation in Chol + buffer from [ 3 H]-TCA accumulation in Na + buffer. Right panel: Relative Na + -dependent [ 3 H]-TCA transport was plotted with respect to everolimus concentration as a non-linear regression to calculate best fit IC 50 of 8.0 ± 1.3 µM. (C) Left panel: Mock-GFP or NTCP-GFP were induced in Flp-In ™ T-REx ™ 293 cells with 1 μg/mL doxycycline for three days. Accumulation of [ 3 H]-TCA (2 μM; 200 nCi/mL) in ECF with Na + over 10 min in the absence (DMSO Ctrl; 0.1%) and presence of everolimus (0.1–100 µM) was measured in stably transfected Mock-GFP and NTCP-GFP-expressing Flp-In ™ T-REx ™ 293 cells. Right panel: Relative [ 3 H]-TCA transport was plotted with respect to everolimus concentration as a non-linear regression to calculate best fit IC 50 of 6.7 ± 1.3 µM. All data were plotted as mean ± SD ( n = 3). Error bars are not visible when the SD is smaller than the data point. (B) and (C) Left panels: Statistically significant differences compared to DMSO Ctrl were measured using an ordinary one-way ANOVA with Dunnett's multiple comparison test (*, p < 0.05, **, p < 0.0001).

Article Snippet: The human hepatoma HuH-7 cell line (JCRB0403) was purchased from Sekisui Xenotech (Kansas City, KS).

Techniques: Concentration Assay, Inhibition, Stable Transfection, Transfection, Expressing

Inhibition of NTCP-mediated taurocholate (TCA) transport by everolimus over time. Accumulation of [ 3 H]-TCA (2 μM; 200 nCi/mL) in extracellular fluid buffer (ECF) with Na + over 5 or 10 min in the absence (DMSO Ctrl; 0.1%) and presence of everolimus (10 µM) was measured in (A) differentiated HuH-7 cells, and (B) stably transfected Mock-GFP and NTCP-GFP-expressing Flp-In ™ T-REx ™ 293 cells. Mock-GFP or NTCP-GFP were induced in Flp-In ™ T-REx ™ 293 cells with 1 μg/mL doxycycline for three days. All data were plotted as mean ± SD ( n = 3). Error bars are not visible when the SD is smaller than the data point.

Journal: Frontiers in Pharmacology

Article Title: Effect of mTOR inhibitors on sodium taurocholate cotransporting polypeptide (NTCP) function in vitro

doi: 10.3389/fphar.2023.1147495

Figure Lengend Snippet: Inhibition of NTCP-mediated taurocholate (TCA) transport by everolimus over time. Accumulation of [ 3 H]-TCA (2 μM; 200 nCi/mL) in extracellular fluid buffer (ECF) with Na + over 5 or 10 min in the absence (DMSO Ctrl; 0.1%) and presence of everolimus (10 µM) was measured in (A) differentiated HuH-7 cells, and (B) stably transfected Mock-GFP and NTCP-GFP-expressing Flp-In ™ T-REx ™ 293 cells. Mock-GFP or NTCP-GFP were induced in Flp-In ™ T-REx ™ 293 cells with 1 μg/mL doxycycline for three days. All data were plotted as mean ± SD ( n = 3). Error bars are not visible when the SD is smaller than the data point.

Article Snippet: The human hepatoma HuH-7 cell line (JCRB0403) was purchased from Sekisui Xenotech (Kansas City, KS).

Techniques: Inhibition, Stable Transfection, Transfection, Expressing

Effect of structural and functional analogs of everolimus on NTCP-mediated taurocholate (TCA) transport. (A) Structures of the mTOR inhibitors tested. Accumulation of [ 3 H]-TCA (2 μM; 200 nCi/mL) in extracellular fluid buffer (ECF) with Na + or without Na + (Chol + ) over 10 min was measured in the presence of 0.1% DMSO control (DMSO Ctrl) or mTOR inhibiting drugs (10 µM) in (B) undifferentiated (HuH-7 Ctrl) and differentiated HuH-7 cells, or (C) Mock-GFP and NTCP-GFP-expressing Flp-In ™ T-REx ™ 293 cells. Mock-GFP or NTCP-GFP were induced in Flp-In ™ T-REx ™ 293 cells with 1 μg/mL doxycycline for three days. All cells were pretreated for 15 min with 0.1% DMSO or drugs (10 µM) in cell culture medium prior to initiation of the accumulation phase. Troglitazone (TGZ; 75 μM), a known inhibitor of NTCP, was used as a positive control. (B) and (C) Left panels: Statistically significant differences compared to DMSO Ctrl were measured using an ordinary two-way ANOVA with Dunnett’s multiple comparison test (*, p < 0.05, **, p < 0.0001). (B) and (C) Right panels: Na + -dependent [ 3 H]-TCA accumulation was calculated by subtracting [ 3 H]-TCA accumulation in Chol + buffer from [ 3 H]-TCA accumulation in Na + buffer. Statistically significant differences compared to DMSO Ctrl were measured using an ordinary one-way ANOVA with Dunnett’s multiple comparison test (*, p < 0.05, **, p < 0.0001). All data were plotted as mean ± SD ( n = 3).

Journal: Frontiers in Pharmacology

Article Title: Effect of mTOR inhibitors on sodium taurocholate cotransporting polypeptide (NTCP) function in vitro

doi: 10.3389/fphar.2023.1147495

Figure Lengend Snippet: Effect of structural and functional analogs of everolimus on NTCP-mediated taurocholate (TCA) transport. (A) Structures of the mTOR inhibitors tested. Accumulation of [ 3 H]-TCA (2 μM; 200 nCi/mL) in extracellular fluid buffer (ECF) with Na + or without Na + (Chol + ) over 10 min was measured in the presence of 0.1% DMSO control (DMSO Ctrl) or mTOR inhibiting drugs (10 µM) in (B) undifferentiated (HuH-7 Ctrl) and differentiated HuH-7 cells, or (C) Mock-GFP and NTCP-GFP-expressing Flp-In ™ T-REx ™ 293 cells. Mock-GFP or NTCP-GFP were induced in Flp-In ™ T-REx ™ 293 cells with 1 μg/mL doxycycline for three days. All cells were pretreated for 15 min with 0.1% DMSO or drugs (10 µM) in cell culture medium prior to initiation of the accumulation phase. Troglitazone (TGZ; 75 μM), a known inhibitor of NTCP, was used as a positive control. (B) and (C) Left panels: Statistically significant differences compared to DMSO Ctrl were measured using an ordinary two-way ANOVA with Dunnett’s multiple comparison test (*, p < 0.05, **, p < 0.0001). (B) and (C) Right panels: Na + -dependent [ 3 H]-TCA accumulation was calculated by subtracting [ 3 H]-TCA accumulation in Chol + buffer from [ 3 H]-TCA accumulation in Na + buffer. Statistically significant differences compared to DMSO Ctrl were measured using an ordinary one-way ANOVA with Dunnett’s multiple comparison test (*, p < 0.05, **, p < 0.0001). All data were plotted as mean ± SD ( n = 3).

Article Snippet: The human hepatoma HuH-7 cell line (JCRB0403) was purchased from Sekisui Xenotech (Kansas City, KS).

Techniques: Functional Assay, Expressing, Cell Culture, Positive Control

Differentiation scheme and the effect of dexamethasone (DEX) and DMSO-mediated differentiation on canalicular formation in HuH-7 cells. ( a ) Schematic depicting HuH-7 differentiation and culture timeline with 1 µM DEX and 0.5% DMSO supplementation and Matrigel overlay. Extended culture was performed for 2 weeks. ( b ) Formation of canalicular-like structures was examined using 5(6)-carboxy-2′,7′-dichlorofluorescein diacetate (CDFDA) in differentiated HuH-7 cells with overlay. Uptake of CDFDA occurs passively into the cell, where intracellular esterase enzymes cleave the diacetate group. The resulting CDF is a fluorescent substrate for apical MRP2-mediated efflux into canalicular-like structures highlighted in green (arrows). Scale bar = 200 µm.

Journal: Scientific Reports

Article Title: A novel differentiated HuH-7 cell model to examine bile acid metabolism, transport and cholestatic hepatotoxicity

doi: 10.1038/s41598-022-18174-z

Figure Lengend Snippet: Differentiation scheme and the effect of dexamethasone (DEX) and DMSO-mediated differentiation on canalicular formation in HuH-7 cells. ( a ) Schematic depicting HuH-7 differentiation and culture timeline with 1 µM DEX and 0.5% DMSO supplementation and Matrigel overlay. Extended culture was performed for 2 weeks. ( b ) Formation of canalicular-like structures was examined using 5(6)-carboxy-2′,7′-dichlorofluorescein diacetate (CDFDA) in differentiated HuH-7 cells with overlay. Uptake of CDFDA occurs passively into the cell, where intracellular esterase enzymes cleave the diacetate group. The resulting CDF is a fluorescent substrate for apical MRP2-mediated efflux into canalicular-like structures highlighted in green (arrows). Scale bar = 200 µm.

Article Snippet: The human hepatoma HuH-7 cell line (JCRB0403) was purchased from Sekisui Xenotech (Kansas City, KS).

Techniques:

$Membrane protein abundance of key hepatic bile acid transporters in control and differentiated HuH-7 cells cultured for 2 weeks with overlay. HuH-7 cells were cultured for 2 weeks without (Ctrl) and with 1 µM dexamethasone (DEX) and/or 0.5% DMSO. ( a ) Abundance of bile acid-relevant transport proteins such as the bile salt export pump (BSEP), sodium taurocholate co-transporting polypeptide (NTCP), organic solute transporter (OST) α, OST β, multidrug resistance-associated protein (MRP) 4, organic anion transporting polypeptide (OATP) 1B3, and Na + /K + ATPase (loading control) was evaluated using Western blotting of membrane fractions harvested from HuH-7 cells with overlay. ( b ) Densitometry was performed using ImageJ and BSEP, NTCP, OSTα, OSTβ, MRP4, and OATP1B3 signals were normalized to Na + /K + ATPase. Relative protein levels were calculated with respect to Ctrl (set to 1.0) and data were plotted as mean ± standard deviation ( n = 3). Statistically significant differences for each protein were assessed using an ordinary one-way ANOVA with Dunnett’s multiple comparison test, compared to control (Ctrl; *, p < 0.05, **, p < 0.0001). N.D., not detected.$

Journal: Scientific Reports

Article Title: A novel differentiated HuH-7 cell model to examine bile acid metabolism, transport and cholestatic hepatotoxicity

doi: 10.1038/s41598-022-18174-z

Figure Lengend Snippet: Membrane protein abundance of key hepatic bile acid transporters in control and differentiated HuH-7 cells cultured for 2 weeks with overlay. HuH-7 cells were cultured for 2 weeks without (Ctrl) and with 1 µM dexamethasone (DEX) and/or 0.5% DMSO. ( a ) Abundance of bile acid-relevant transport proteins such as the bile salt export pump (BSEP), sodium taurocholate co-transporting polypeptide (NTCP), organic solute transporter (OST) α, OST β, multidrug resistance-associated protein (MRP) 4, organic anion transporting polypeptide (OATP) 1B3, and Na + /K + ATPase (loading control) was evaluated using Western blotting of membrane fractions harvested from HuH-7 cells with overlay. ( b ) Densitometry was performed using ImageJ and BSEP, NTCP, OSTα, OSTβ, MRP4, and OATP1B3 signals were normalized to Na + /K + ATPase. Relative protein levels were calculated with respect to Ctrl (set to 1.0) and data were plotted as mean ± standard deviation ( n = 3). Statistically significant differences for each protein were assessed using an ordinary one-way ANOVA with Dunnett’s multiple comparison test, compared to control (Ctrl; *, p < 0.05, **, p < 0.0001). N.D., not detected.

Article Snippet: The human hepatoma HuH-7 cell line (JCRB0403) was purchased from Sekisui Xenotech (Kansas City, KS).

Techniques: Cell Culture, Western Blot, Standard Deviation

Abundance of bile acid-relevant proteins in differentiated HuH-7 cells with overlay measured by quantitative targeted absolute proteomics (QTAP) analysis. Membrane protein abundance (pmol/mg protein; mean ± standard deviation, n = 3) of select bile acid-relevant proteins in 2-week cultured HuH-7 cells treated with 1 µM dexamethasone (DEX) and/or 0.5% DMSO was plotted as scatter plots for individual proteins. Statistically significant differences with respect to control (Ctrl) were calculated for each protein using an ordinary one-way ANOVA and Dunnett’s multiple comparison test (*, p < 0.05). N.D., not detected; LLOQ, lower limit of quantitation (0.1 pmol/mg protein).

Journal: Scientific Reports

Article Title: A novel differentiated HuH-7 cell model to examine bile acid metabolism, transport and cholestatic hepatotoxicity

doi: 10.1038/s41598-022-18174-z

Figure Lengend Snippet: Abundance of bile acid-relevant proteins in differentiated HuH-7 cells with overlay measured by quantitative targeted absolute proteomics (QTAP) analysis. Membrane protein abundance (pmol/mg protein; mean ± standard deviation, n = 3) of select bile acid-relevant proteins in 2-week cultured HuH-7 cells treated with 1 µM dexamethasone (DEX) and/or 0.5% DMSO was plotted as scatter plots for individual proteins. Statistically significant differences with respect to control (Ctrl) were calculated for each protein using an ordinary one-way ANOVA and Dunnett’s multiple comparison test (*, p < 0.05). N.D., not detected; LLOQ, lower limit of quantitation (0.1 pmol/mg protein).

Article Snippet: The human hepatoma HuH-7 cell line (JCRB0403) was purchased from Sekisui Xenotech (Kansas City, KS).

Techniques: Standard Deviation, Cell Culture, Quantitation Assay

Cellular localization of bile acid transporters in control and differentiated HuH-7 cells with overlay. Immunofluorescence and confocal microscopy were performed to examine transporter localization in control and differentiated [culture medium contained 1 µM dexamethasone (DEX) + 0.5% DMSO] HuH-7 cells with overlay. Immunostaining of zonula occludens 1 (ZO1) and Na + /K + ATPase were used as markers for the canalicular and basolateral membranes, respectively. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI). Localization of the following bile acid transport proteins was determined in control and differentiated HuH-7 cells ( a ) bile salt export pump (BSEP) and multidrug resistance-associated protein (MRP) 2, ( b ) MRP3 and MRP4 (white arrow indicates membrane localized protein), ( c ) sodium taurocholate co-transporting polypeptide (NTCP), ( d ) organic anion transporting polypeptide (OATP) 1B1, OATP1B3, and OATP2B1, and ( e ) organic solute transporter (OST) α and OST β. At least three areas were randomly imaged for each sample. All projected images were analyzed using ImageJ software. Scale bar = 20 µm.

Journal: Scientific Reports

Article Title: A novel differentiated HuH-7 cell model to examine bile acid metabolism, transport and cholestatic hepatotoxicity

doi: 10.1038/s41598-022-18174-z

Figure Lengend Snippet: Cellular localization of bile acid transporters in control and differentiated HuH-7 cells with overlay. Immunofluorescence and confocal microscopy were performed to examine transporter localization in control and differentiated [culture medium contained 1 µM dexamethasone (DEX) + 0.5% DMSO] HuH-7 cells with overlay. Immunostaining of zonula occludens 1 (ZO1) and Na + /K + ATPase were used as markers for the canalicular and basolateral membranes, respectively. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI). Localization of the following bile acid transport proteins was determined in control and differentiated HuH-7 cells ( a ) bile salt export pump (BSEP) and multidrug resistance-associated protein (MRP) 2, ( b ) MRP3 and MRP4 (white arrow indicates membrane localized protein), ( c ) sodium taurocholate co-transporting polypeptide (NTCP), ( d ) organic anion transporting polypeptide (OATP) 1B1, OATP1B3, and OATP2B1, and ( e ) organic solute transporter (OST) α and OST β. At least three areas were randomly imaged for each sample. All projected images were analyzed using ImageJ software. Scale bar = 20 µm.

Article Snippet: The human hepatoma HuH-7 cell line (JCRB0403) was purchased from Sekisui Xenotech (Kansas City, KS).

Techniques: Immunofluorescence, Confocal Microscopy, Immunostaining, Staining, Software

Bile acid profiling and gene expression analysis in HuH-7 cells with overlay. ( a ) Cellular bile acids were measured in overlaid HuH-7 cells cultured for 2 weeks without (Ctrl) and with 1 µM dexamethasone (DEX) and/or 0.5% DMSO using liquid chromatography with tandem mass spectrometry (LC–MS/MS). HuH-7 cells were incubated in standard Hanks' balanced salt solution (HBSS; With Ca 2+ ) or Ca 2+ -free HBSS buffer (Without Ca 2+ ) for 10 min at 37˚C prior to analysis for determination of “Cells + Bile” and “Cells” content, respectively. Average total bile acid species were plotted based on HuH-7 cell culture conditions. Concentrations of each bile acid species [cholic acid (CA), chenodeoxycholic acid (CDCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), GCDCA 3-sulfate (GCDCA-S), taurocholic acid (TCA), and taurochenodeoxycholic acid (TCDCA)] were calculated, normalized to total protein, and plotted as mean ± standard deviation ( n = 3) based on HuH-7 cell culture conditions. In all cases, the biliary excretion index (BEI) was calculated using Eq. , and BEI values (%) were included above the relevant data in the graphs, when measurable. The lower limit of quantitation was 0.1 nM for GCA, TCA and TCDCA, 0.5 nM for GCDCA and CA, and 1 nM for CDCA and GCDCA-S. ( b ) mRNA was measured by RT-qPCR in HuH-7 cells cultured for 2 weeks with overlay, 1 µM DEX and/or 0.5% DMSO. Threshold cycle (C T ) values of each gene of interest [ cytochrome P450 ( CYP) 7A1 , CYP8B1 , CYP27A1, bile acid-CoA: amino acid N-acyltransferase (BAAT), bile acid-CoA synthase (BACS), CYP7B1, and CYP3A4 ] were normalized to the housekeeping gene β-actin ( ACTB ) and compared with control (Ctrl; set to 1.0). Data are plotted as mean ± standard deviation (n = 3). Statistically significant differences in relative mRNA were determined by an ordinary one-way ANOVA with Dunnett’s multiple comparison test (* p -value < 0.05, ** < 0.0001, DEX, DMSO or DEX + DMSO vs. Ctrl).

Journal: Scientific Reports

Article Title: A novel differentiated HuH-7 cell model to examine bile acid metabolism, transport and cholestatic hepatotoxicity

doi: 10.1038/s41598-022-18174-z

Figure Lengend Snippet: Bile acid profiling and gene expression analysis in HuH-7 cells with overlay. ( a ) Cellular bile acids were measured in overlaid HuH-7 cells cultured for 2 weeks without (Ctrl) and with 1 µM dexamethasone (DEX) and/or 0.5% DMSO using liquid chromatography with tandem mass spectrometry (LC–MS/MS). HuH-7 cells were incubated in standard Hanks' balanced salt solution (HBSS; With Ca 2+ ) or Ca 2+ -free HBSS buffer (Without Ca 2+ ) for 10 min at 37˚C prior to analysis for determination of “Cells + Bile” and “Cells” content, respectively. Average total bile acid species were plotted based on HuH-7 cell culture conditions. Concentrations of each bile acid species [cholic acid (CA), chenodeoxycholic acid (CDCA), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), GCDCA 3-sulfate (GCDCA-S), taurocholic acid (TCA), and taurochenodeoxycholic acid (TCDCA)] were calculated, normalized to total protein, and plotted as mean ± standard deviation ( n = 3) based on HuH-7 cell culture conditions. In all cases, the biliary excretion index (BEI) was calculated using Eq. , and BEI values (%) were included above the relevant data in the graphs, when measurable. The lower limit of quantitation was 0.1 nM for GCA, TCA and TCDCA, 0.5 nM for GCDCA and CA, and 1 nM for CDCA and GCDCA-S. ( b ) mRNA was measured by RT-qPCR in HuH-7 cells cultured for 2 weeks with overlay, 1 µM DEX and/or 0.5% DMSO. Threshold cycle (C T ) values of each gene of interest [ cytochrome P450 ( CYP) 7A1 , CYP8B1 , CYP27A1, bile acid-CoA: amino acid N-acyltransferase (BAAT), bile acid-CoA synthase (BACS), CYP7B1, and CYP3A4 ] were normalized to the housekeeping gene β-actin ( ACTB ) and compared with control (Ctrl; set to 1.0). Data are plotted as mean ± standard deviation (n = 3). Statistically significant differences in relative mRNA were determined by an ordinary one-way ANOVA with Dunnett’s multiple comparison test (* p -value < 0.05, ** < 0.0001, DEX, DMSO or DEX + DMSO vs. Ctrl).

Article Snippet: The human hepatoma HuH-7 cell line (JCRB0403) was purchased from Sekisui Xenotech (Kansas City, KS).

Techniques: Expressing, Cell Culture, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Incubation, Standard Deviation, Quantitation Assay, Quantitative RT-PCR

Effect of dexamethasone (DEX) and DMSO on bile acid uptake and biliary excretion index (BEI) in differentiated HuH-7 cells. ( a ) Uptake of 4 µM taurocholic acid ([ 3 H]-TCA; 400 nCi/mL) was measured in control (Ctrl) and differentiated (1 µM DEX + 0.5% DMSO) HuH-7 cells without overlay using extracellular fluid (ECF; N = 2, in triplicate) buffer over 10 min. ( b ) Accumulation and BEI of 2 µM [ 3 H]-TCA (200 nCi/mL) were measured in HuH-7 cells with overlay in standard or Ca 2+ -free Hanks balanced salt solution buffer (HBSS) at various DMSO concentrations, and ( c ) in the presence and absence of DEX, overlay and 0.5% DMSO. Data were plotted as mean ± standard deviation ( n = 3) and BEI was calculated using Eq. . Statistically significant differences were measured using an ordinary two-way ANOVA with Dunnett’s multiple comparisons test [*, p < 0.05, **, p < 0.0001, ( b ) cells cultured with overlay and with DMSO vs. without DMSO; ( c ) control cells cultured without vs with overlay and cells cultured with overlay with DEX ± DMSO]. @ Cell detachment observed.

Journal: Scientific Reports

Article Title: A novel differentiated HuH-7 cell model to examine bile acid metabolism, transport and cholestatic hepatotoxicity

doi: 10.1038/s41598-022-18174-z

Figure Lengend Snippet: Effect of dexamethasone (DEX) and DMSO on bile acid uptake and biliary excretion index (BEI) in differentiated HuH-7 cells. ( a ) Uptake of 4 µM taurocholic acid ([ 3 H]-TCA; 400 nCi/mL) was measured in control (Ctrl) and differentiated (1 µM DEX + 0.5% DMSO) HuH-7 cells without overlay using extracellular fluid (ECF; N = 2, in triplicate) buffer over 10 min. ( b ) Accumulation and BEI of 2 µM [ 3 H]-TCA (200 nCi/mL) were measured in HuH-7 cells with overlay in standard or Ca 2+ -free Hanks balanced salt solution buffer (HBSS) at various DMSO concentrations, and ( c ) in the presence and absence of DEX, overlay and 0.5% DMSO. Data were plotted as mean ± standard deviation ( n = 3) and BEI was calculated using Eq. . Statistically significant differences were measured using an ordinary two-way ANOVA with Dunnett’s multiple comparisons test [*, p < 0.05, **, p < 0.0001, ( b ) cells cultured with overlay and with DMSO vs. without DMSO; ( c ) control cells cultured without vs with overlay and cells cultured with overlay with DEX ± DMSO]. @ Cell detachment observed.

Article Snippet: The human hepatoma HuH-7 cell line (JCRB0403) was purchased from Sekisui Xenotech (Kansas City, KS).

Techniques: Standard Deviation, Cell Culture

Effect of cholestatic drugs on the accumulation and biliary excretion index (BEI) of taurocholate (TCA) in control and differentiated HuH-7 cells with overlay. Accumulation and BEI of 2 µM [ 3 H]-TCA (200 nCi/mL) were measured in control (untreated; Ctrl) and differentiated (1 µM DEX + 0.5% DMSO) HuH-7 cells with overlay in standard or Ca 2+ -free HBSS buffer after a 24-h incubation with cholestatic drugs. On day 13 of culture, differentiated HuH-7 cells were exposed to 0.1% DMSO control (vehicle), ritonavir (RTV; 25 µM), pioglitazone (PIO; 100 µM), dasatinib (DAS; 20 µM), or troglitazone (TGZ; 75 µM) for 24 h. Data were plotted as mean ± standard deviation ( n = 3) and biliary excretion index (BEI, %) was calculated using Eq. . Statistically significant differences were measured using an ordinary two-way ANOVA with Dunnett’s multiple comparisons test (*, p < 0.05, **, p < 0.0001, vehicle control vs. drug treatment).

Journal: Scientific Reports

Article Title: A novel differentiated HuH-7 cell model to examine bile acid metabolism, transport and cholestatic hepatotoxicity

doi: 10.1038/s41598-022-18174-z

Figure Lengend Snippet: Effect of cholestatic drugs on the accumulation and biliary excretion index (BEI) of taurocholate (TCA) in control and differentiated HuH-7 cells with overlay. Accumulation and BEI of 2 µM [ 3 H]-TCA (200 nCi/mL) were measured in control (untreated; Ctrl) and differentiated (1 µM DEX + 0.5% DMSO) HuH-7 cells with overlay in standard or Ca 2+ -free HBSS buffer after a 24-h incubation with cholestatic drugs. On day 13 of culture, differentiated HuH-7 cells were exposed to 0.1% DMSO control (vehicle), ritonavir (RTV; 25 µM), pioglitazone (PIO; 100 µM), dasatinib (DAS; 20 µM), or troglitazone (TGZ; 75 µM) for 24 h. Data were plotted as mean ± standard deviation ( n = 3) and biliary excretion index (BEI, %) was calculated using Eq. . Statistically significant differences were measured using an ordinary two-way ANOVA with Dunnett’s multiple comparisons test (*, p < 0.05, **, p < 0.0001, vehicle control vs. drug treatment).

Article Snippet: The human hepatoma HuH-7 cell line (JCRB0403) was purchased from Sekisui Xenotech (Kansas City, KS).

Techniques: Incubation, Standard Deviation

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