10 research outputs found

    ERRγ inverse agonist GSK5182 down-regulates the hypoxia-induced PDK4 expression.

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    <p>(<b>A–B</b>) HepG2 cells were transfected with hPDK4-Luc. After transfection, the cells were exposed in hypoxia and treated with or without GSK5182. Harvested lysates were utilized for luciferase and β-galactosidase assay (A). Q-PCR was performed using isolated total RNA (B). Experiments were done in triplicate and data are expressed as the fold activation relative to the control. (<b>C</b>) HepG2 cells were seeded in 60-cm<sup>2</sup> dishes and incubated overnight. The cells were incubated in hypoxia and treated with or without GSK5182. Total protein was harvesed for Western blot analysis using indicated antibodies. (<b>D</b>) HepG2 cells were seeded in 60-cm<sup>2</sup> dishes and incubated overnight. The cells were treated with or without chemicals (DFO and GSK5182) for 6 hr. Total protein and mRNA were isolated for Western blot assay and RT-PCR and normalized with α or β-tubulin and β-actin. (<b>E</b>) A schematic representation. All data are representative of at least three independent experiments. Error bars show ± S.E.M. <sup>**</sup><i>P</i><0.01, <sup>***</sup><i>P</i><0.001 by two-tailed Student <i>t</i>-test.</p

    Hypoxia induces the ERRγ gene expression in hepatoma cell lines.

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    <p>(<b>A–B</b>), HepG2 cells were seeded in 60-cm<sup>2</sup> dishes and incubated overnight. Then cells were incubated under hypoxia at indicated time period. The expression of ERRγ was analyzed by Western blot (<b>A</b>) and Q-PCR (<b>B</b>) analysis. (<b>C–D</b>) HepG2 cells were seeded in 60-cm<sup>2</sup> dishes and incubated overnight and then treated with DFO at indicated concentration and time period. The expression of ERRs was analyzed by Western blot (<b>C</b>) and Q-PCR (<b>D</b>) analysis. ERRγ gene expression was normalized to L32 gene expression, and α or β-tubulin expression. All data are representative of at least three independent experiments. Error bars show ± S.E.M. <sup>*</sup><i>P</i><0.05, <sup>**</sup><i>P</i><0.01 by two-tailed Student <i>t</i>-test.</p

    HIF-1α increases the expression of ERR γ.

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    <p>(<b>A</b>) HepG2 cells were transfected with nonspecific siRNA (NS) or si-HIF-1α, and isolated total protein was analyzed by Western blot. α-tubulin was used as a control. (<b>B</b>) HepG2 cells were transfected with pcDNA3-HIF-1α, pcDNA3-ARNT and hERRγ-Luc, respectively. Experiments were conducted in duplicate and data are expressed as the fold activation relative to the control. (<b>C</b>) HepG2 cells were transfected with pcDNA3-HIF-1α and pcDNA3-ARNT and Q-PCR was performed using isolated total RNA. (<b>D–E</b>) HepG2 cells were transfected with nonspecific siRNA (NS) or si-HIF-1α. After transfection, lysates were utilized for luciferase and β-galactosidase assay (D). Q-PCR was performed using isolated total RNA from HepG2 cells (E). All data are representative of at least three independent experiments. Error bars show ± S.E.M. <sup>*</sup><i>P</i><0.05, <sup>***</sup><i>P</i><0.001 by two-tailed Student <i>t</i>-test.</p

    Hypoxic activation of HIF-1α directly regulates the transcriptional activity of ERRγ.

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    <p>(<b>A</b>) HepG2 cells were transfected with hERRγ-Luc. After 24 h of the transfection, HepG2 cells were exposed to hypoxia for indicated time period. Experiments were carried out in triplicate and data are expressed as the fold activation relative to the control. (<b>B–C</b>) HepG2 cells were transfected with hERRγ (−2 kb)-Luc, hERRγ (−1 kb)-Luc, hERRγ (−0.5 kb)-Luc, hERRγ (−0.3 kb)-Luc, hERRγ (HREmt1)-Luc, hERRγ (HREmt2)-Luc, hERRγ (HREmt1+2)-Luc. After 24 h of the transfection, HepG2 cells were exposed to hypoxia for 9 hr and analyzed using luciferase and β-galactosidase assay. Experiments were performed in duplicate and data are expressed as the fold activation relative to the control. (<b>D</b>) ChIP assay: HepG2 cell was exposed to hypoxia for 9 hr. Input represents 10% of purified DNA in each sample. Cell extracts were immunoprecipitated with anti-HIF-1α and purified DNA samples were employed for Q-PCR with primers binding to HRE1 (−1080 to −849) and HRE2 (−508 to −295) and distal site (−1826 to −1586) on the <i>ERRγ</i> gene promoter. All data are representative of at least three independent experiments. Error bars show ± S.E.M. <sup>***</sup><i>P</i><0.001 by two-tailed Student <i>t</i>-test.</p

    ERRγ directly regulates hypoxia mediated PDK4 gene expression.

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    <p>(<b>A</b>) HepG2 cells were transfected with hPDK4-Luc. After transfection, the cells were exposed to hypoxia for indicated period and lysates were utilized for luciferase and β-galactosidase assay. Experiments were done in triplicate and data are expressed as the fold activation relative to the control. (<b>B</b>) HepG2 cells were seeded in 60-cm<sup>2</sup> dishes and trasnfected siERRγ and control siRNA for 72 hr and then exposed hypoxia for 9 hr. Cells were harvested for analyzing luciferase and β-galactodidase assay. (<b>C–D</b>) HepG2 cells were transfected with several deletion constructs of hPDK4 (−848)-Luc, hPDK4 (−500)-Luc, hPDK4 (−291)-Luc and hPDK4-mtERRE1-Luc with pcDNA3-ERRγ in the presence or absence with hypoxia exposure, respectively. 48 hr after transfection, the cells were harvested and performed luciferase and β-galactodidase assay. Experiments were done in duplicate and data expressed as the fold activation related to control. (<b>E</b>) HepG2 cell were transiently transfected with hPDK4 (−848)-Luc, hPDK4-mtERRE1-Luc, pcDNA3-ERRγ and pcDNA3-HIF-1α. (<b>F</b>) ChIP assay: HepG2 cell was exposed to hypoxia for 9 hr. Input represents 10% of purified DNA in each sample. Cell extracts were immunoprecipitated with anti-ERRα and purified DNA samples were employed for Q-PCR with primers binding to ERRE (−502 to −252) and distal site (−1056 to −886) on the <i>PDK4</i> gene promoter. All data are representative of at least three independent experiments. Error bars show ± S.E.M. <sup>***</sup><i>P</i><0.001 by two-tailed Student <i>t</i>-test.</p

    ERRγ mediates the hypoxia induced expression of PDK4.

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    <p>(<b>A–B</b>) HepG2 cells were seeded in 60-cm<sup>2</sup> dishes and exposed to hypoxia for indicated time period. Total RNA and protein were isolated and used for Western blot (<b>A</b>) and Q-PCR (<b>B</b>), respectively. (<b>C–D</b>) HepG2 was treated with DFO for indicated concentration and time period and then cells were harvested. Total protein was harvested for Western blot (<b>C</b>) using indicated antibodies and was normalized to β-tubulin expression. And total RNA was isolated for Q-PCR (<b>D</b>). The mRNA levels of PDK2 and PDK4 were normalized to L32 gene expression. (<b>E</b>) Effect of knockdown of ERRγ. HepG2 cells were infected with Ad-US and Ad- shERRγ for 48 hr, respectively. Total protein was isolated for Western blot analysis of PDK4 and then was normalized to β-tubulin or α-tubulin expression. All data are representative of at least three independent experiments. Error bars show ± S.E.M. <sup>*</sup><i>P</i><0.05 by two-tailed Student <i>t</i>-test.</p

    ACEA induces <i>FGF21</i> gene expression.

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    <p>(A–C) HepG2 cells, rat primary hepatocytes (RPH), and AML12 cells were treated with ACEA (10 μM) for the indicated time periods. (D) Wild-type or CB1<sup>-/-</sup> mouse primary hepatocytes (MPH) were treated with ACEA (10 μM) for 3 h. (E) Mice were treated with ACEA (10 mg/kg) for the indicated number of days. Livers were harvested for mRNA analysis. (A–E) <i>FGF21</i> and <i>ERRγ</i> mRNA levels were measured by quantitative qPCR analysis and normalized to <i>actin</i> mRNA levels. All data are the means ± standard errors of at least three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 by one-way ANOVA.</p

    GSK5182 inhibits ACEA-mediated induction of <i>FGF21</i> gene expression.

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    <p>(A) AML12 cells were transfected with mFGF21-Luc and treated with ACEA (10 μM) for 3 h with or without GSK5182 (10 μM). (B–D) HepG2 cells, AML12 cells, and mouse primary hepatocytes (MPH) were treated with ACEA (10 μM) for 3 h with or without GSK5182 (10 μM). (E and G) GSK5182 (40 mg/kg) was administrated to male C57BL/6J mice (n = 3–4 per group) daily by intraperitoneal injection for 4 days. ACEA (10 mg/kg) was also given by intraperitoneal injection daily during the final 3 days. (A–D) <i>FGF21</i> and <i>ERRγ</i> mRNA levels were measured by qPCR analysis and normalized to <i>actin</i> mRNA levels. (F) AML12 cells were treated with ACEA (10 μM) for 3 h with or without GSK5182 (10 μM). Culture media was recovered for FGF21 secretion analysis. (G) Male C57BL/6J mice (n = 3) were treated with ACEA (10 mg/kg) with and without GSK5182 (40 mg/kg) daily for 3 days. Serum was analyzed for FGF21 secretion. (H) Male C57BL/6J mice (n = 5 per group) were fed an alcohol-containing diet for 4 weeks and GSK5182 (40mg/kg once daily) was given by oral gavage for the final 2 weeks of alcohol feeding. (I) Schematic diagram of ERRγ-mediated <i>FGF21</i> gene expression. GSK5182 inhibits activation of <i>FGF21</i> gene expression and FGF21 secretion mediated by increased ERRγ caused by activation of the hepatic CB1 receptor. All data are the means ± standard errors for at least three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 by one-way ANOVA.</p

    ERRγ overexpression induces <i>FGF21</i> gene expression.

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    <p>(A–C) HepG2 cells, AML12 cells, and mouse primary hepatocytes (MPH) were infected with Ad-GFP and Ad-ERRγ. (D) Ad-GFP or Ad-ERRγ was injected into male C57BL/6J mice via the tail vein. Mice were sacrificed at 5 days after injection. (A–D) <i>FGF21</i> and <i>ERRγ</i> mRNA levels were measured by quantitative qPCR analysis and normalized to <i>actin</i> mRNA levels. (E) Culture media of adenovirus-infected AML12 cells was obtained for FGF21 secretion analysis. (F) Ad-GFP or Ad-ERRγ was injected via the tail vein into male C57BL/6J mice. Serum from these mice was analyzed for FGF21 secretion. All data are the means ± standard errors of at least three independent experiments. ***p < 0.001 by Student’s t-test.</p

    ACEA increases FGF21 protein levels.

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    <p>(A–C) Whole cell lysates of ACEA-treated HepG2 cells and AML12 cells and livers of ACEA-treated intact mice were harvested for western blot analysis. (D–E) AML12 cells and rat primary hepatocytes were treated with ACEA (10 μM) for the indicated time periods. Culture media were collected for FGF21 secretion analysis. (F) Mice were treated with ACEA (10 mg/kg) for the indicated number of days. Serum was obtained for FGF21 secretion analysis. All data are the means ± standard errors of at least three independent experiments. **p < 0.01; ***p < 0.001 by one-way ANOVA.</p
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